Poly(ethylene glycol) containing chemically disparate endgroups

ABSTRACT

The present invention provides bifunctional polymers, methods of preparing the same, and intermediates thereto. These compounds are useful in a variety of applications including the PEGylation of biologically active molecules. The invention also provides methods of using said compounds and compositions thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/795,412, filed Apr. 27, 2006, the entirety of which ishereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of polymer chemistry and moreparticularly to functionalized polymers, uses thereof, and intermediatesthereto.

BACKGROUND OF THE INVENTION

Poly(ethylene glycol), also known as PEG, is useful in a variety oftechnological areas and is generally known by the formulaHO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH, wherein n typically ranges fromabout 3 to about 4000. In particular, there is great interest inutilizing PEG, and derivatives thereof, in the pharmaceutical andbiomedical fields. This interest stems from the fact that PEG isnontoxic, biocompatible, non-immunogenic, soluble in water and othersolvents, and is amenable to a variety of therapeutic applicationsincluding pharmaceutical formulations and drug delivery systems, amongothers.

One such area of interest relates to “PEGylation” or “conjugation” whichrefers to the modification of other molecules, especially biomolecules,using PEG and derivatives thereof. PEGylation is often utilized in orderto impart the desirable characteristics of PEG to a particular moleculeor biological scaffold. Such molecules or scaffolds targeted forPEGylation include proteins, dyes, peptides, hydrogels, cells, viruses,and drugs, to name but a few. In the case of drugs, the formation ofPEG-drug conjugates is also of interest to improve aqueous solubility ofhydrophobic drugs and improve biodistribution profiles. In addition, PEGhas been utilized with a variety of natural and synthetic substratesincluding biological implants, medical devices, and the like.Accordingly, it would be advantageous to provide heterobifunctionalizedPEG's having a variety of terminal functional groups.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. GeneralDescription of the Invention

In certain embodiments, the present invention provides a compound offormula I:

or a salt thereof, wherein:

-   n is 10-2500;-   R¹ and R² are each independently hydrogen, halogen, NO₂, CN, N₃,    —N═C═O, —C(R)═NN(R)₂, —P(O)(OR)₂, —P(O)(X)₂, a 9-30 membered crown    ether, or an optionally substituted group selected from aliphatic, a    3-8 membered saturated, partially unsaturated, or aryl ring having    0-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, an 8-10 membered saturated, partially unsaturated, or aryl    bicyclic ring having 0-5 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or a detectable moiety;-   each X is independently halogen;-   each R is independently hydrogen or an optionally substituted    selected from aliphatic or a 3-8 membered, saturated, partially    unsaturated, or aryl ring having 0-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur; and-   L¹ and L² are each independently a valence bond or a bivalent,    saturated or unsaturated, straight or branched C₁₋₁₂ hydrocarbon    chain, wherein 0-6 methylene units of L¹ and L² are independently    replaced by -Cy-, —O—, —NR—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—,    —SO₂—, —NRSO₂—, —SO₂NR—, —NRC(O)—, —C(O)NR—, —OC(O)NR—, or    —NRC(O)O—, wherein:    -   each -Cy- is independently an optionally substituted 3-8        membered bivalent, saturated, partially unsaturated, or aryl        ring having 0-4 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, or an optionally substituted 8-10        membered bivalent saturated, partially unsaturated, or aryl        bicyclic ring having 0-5 heteroatoms independently selected from        nitrogen, oxygen, or sulfur.

2. Definitions

Compounds of this invention include those described generally above, andare further illustrated by the embodiments, sub-embodiments, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As used herein, the phrase “living polymer chain-end” refers to theterminus resulting from a polymerization reaction which maintains theability to react further with additional monomer or with apolymerization terminator.

As used herein, the term “termination” refers to attaching a terminalgroup to a living polymer chain-end by reacting the living polymerchain-end with a polymerization terminator. Alternatively, the term“termination” may refer to the attachment of a terminal group to ahydroxyl end, or derivative thereof, of the polymer chain.

As used herein, the term “polymerization terminator” is usedinterchangeably with the term “polymerization terminating agent” andrefers to compounds that react with a living polymer chain-end to afforda polymer with a terminal group. Alternatively, the term “polymerizationterminator” may refer to a compound that may react with a hydroxyl end,or derivative thereof, of the polymer chain to afford a polymer with aterminal group.

As used herein, the term “polymerization initiator” refers to acompound, or anion thereof, which reacts with ethylene oxide in a mannerwhich results in polymerization thereof. In certain embodiments, thepolymerization initiator is the anion of a functional group whichinitiates the polymerization of ethylene oxide.

The term “aliphatic” or “aliphatic group”, as used herein, denotes ahydrocarbon moiety that may be straight-chain (i.e., unbranched),branched, or cyclic (including fused, bridging, and spiro-fusedpolycyclic) and may be completely saturated or may contain one or moreunits of unsaturation, but which is not aromatic. Unless otherwisespecified, aliphatic groups contain 1-20 carbon atoms. In someembodiments, aliphatic groups contain 1-10 carbon atoms. In otherembodiments, aliphatic groups contain 1-8 carbon atoms. In still otherembodiments, aliphatic groups contain 1-6 carbon atoms, and in yet otherembodiments aliphatic groups contain 1-4 carbon atoms. Suitablealiphatic groups include, but are not limited to, linear or branched,alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon. This includes any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen, or; a substitutable nitrogen of a heterocyclic ring including═N— as in 3,4-dihydro-2H-pyrrolyl, —NH— as in pyrrolidinyl, or═N(R^(†))— as in N-substituted pyrrolidinyl.

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent, saturated or unsaturated, straightor branched C₁₋₁₂ hydrocarbon chain”, refers to bivalent alkylene,alkenylene, and alkynylene chains that are straight or branched asdefined herein.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains three to seven ring members.The term “aryl” may be used interchangeably with the term “aryl ring”.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(◯); —(CH₂)₀₋₄OR^(◯); —O—(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄CH(OR^(◯))₂; —(CH₂)₀₋₄SR^(◯); —(CH₂)₀₋₄Ph, which may besubstituted with R^(◯); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(◯); —CH═CHPh, which may be substituted with R^(◯); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(◯) ₂; —(CH₂)₀₋₄N(R^(◯))C(O)R^(◯); —N(R^(◯))C(S)R^(◯);—(CH₂)₀₋₄N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯))C(S)NR^(◯) ₂;—(CH₂)₀₋₄N(R^(◯))C(O)OR^(◯); —N(R^(◯)N(R^(◯))C(O)R^(◯);—N(R^(◯)N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯)N(R^(◯))C(O)OR^(◯);—(CH₂)₀₋₄C(O)R^(◯); —C(S)R^(◯); —(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄C(O)SR^(◯); —(CH₂)₀₋₄C(O)OsiR^(◯) ₃; —(CH₂)₀₋₄OC(O)R^(◯);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(◯); —(CH₂)₀₋₄SC(O)R^(◯); —(CH₂)₀₋₄C(O)NR^(◯)₂; —C(S)NR^(◯) ₂; —C(S)SR^(◯); —SC(S)SR^(◯), —(CH₂)₀₋₄OC(O)NR^(◯) ₂;—C(O)N(OR^(◯))R^(◯); —C(O)C(O)R^(◯); —C(O)CH₂C(O)R^(◯);—C(NOR^(◯))R^(◯); —(CH₂)₀₋₄ SSR^(◯); —(CH₂)₀₋₄S(O)₂R^(◯);—(CH₂)₀₋₄(O)₂OR^(◯); —(CH₂)₀₋₄OS(O)₂R^(◯); —S(O)₂NR^(◯) ₂;—(CH₂)₀₋₄(O)R^(◯); —N(R^(◯)S(O)₂NR^(◯) ₂; —N(R^(◯)S(O)₂R^(◯);—N(OR^(◯)R^(◯); —C(NH)NR^(◯) ₂; —P(O)₂R^(◯); —P(O)R^(◯) ₂; —OP(O)R^(◯)₂; —OP(O)(OR^(◯))₂; SiR^(◯) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(◯) ₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(◯)) ₂, wherein each R^(◯) may besubstituted as defined below and is independently hydrogen, C₁₋₆aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or, notwithstanding the definitionabove, two independent occurrences of R^(◯), taken together with theirintervening atom(s), form a 3-12-membered saturated, partiallyunsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, which may besubstituted as defined below.

Suitable monovalent substituents on R^(◯) (or the ring formed by takingtwo independent occurrences of R^(◯) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(), —O(halo R^()),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(), —(CH₂)₀₋₂CH(OR^())₂; —O(haloR^()), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(),—(CH₂)₀₋₂SR^(), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NH R^(),—(CH₂)₀₋₂NR^() ₂, —NO₂, —SiR^() ₃, —OSiR^() ₃, —C(O)SR^(), —(C₁₋₄straight or branched alkylene)C(O)OR^(), or —SSR^() wherein each R^()is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(◯) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. A suitable tetravalent substituentthat is bound to vicinal substitutable methylene carbons of an“optionally substituted” group is the dicobalt hexacarbonyl clusterrepresented by

when depicted with the methylenes which bear it.

Suitable substituents on the aliphatic group of R* include halogen,—R^(), —O(haloR^()), —OH, —OR^(), —O(haloR^()), —CN, —C(O)OH,—C(O)OR^(), —NH₂, —NHR^(), —NR^() ₂, or —NO₂, wherein each R^() isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(), —(haloR^(), —OH, —OR^(), —O(haloR^()), —CN, —C(O)OH,—C(O)OR^(), —NH₂, —NHR^(), —NR^() ₂, or —NO₂, wherein each R^() isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benzylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl ethers includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Alkoxyalkyl ethers include acetals such asmethoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethersinclude benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl,O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,p-cyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Suitable mono-protected amines furtherinclude, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected aminomoieties include t-butyloxycarbonylamino (-NHBOC),ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxycarbonylamino, allyloxycarbonylamino(-NHAIloc),benzyloxocarbonylamino (-NHCBZ), allylamino, benzylamino(-NHBn),fluorenylmethylcarbonyl(-NHFmoc), formamido, acetamido, chloroacetamido,dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like.Suitable di-protected amines include amines that are substituted withtwo substituents independently selected from those described above asmono-protected amines, and further include cyclic imides, such asphthalimide, maleimide, succinimide, and the like. Suitable di-protectedamines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected aldehydesfurther include, but are not limited to, acyclic acetals, cyclicacetals, hydrazones, imines, and the like. Examples of such groupsinclude dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzylacetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes,semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected carboxylicacids further include, but are not limited to, optionally substitutedC₁₋₆ aliphatic esters, optionally substituted aryl esters, silyl esters,activated esters, amides, hydrazides, and the like. Examples of suchester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,benzyl, and phenyl ester, wherein each group is optionally substituted.Additional suitable protected carboxylic acids include oxazolines andortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Suitable protected thiols further include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, and thiocarbamates, and the like. Examplesof such groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

A “crown ether moiety” is the radical of a crown ether. A crown ether isa monocyclic polyether comprised of repeating units of —CH₂CH₂O—.Examples of crown ethers include 12-crown-4,15-crown-5, and 18-crown-6.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and relates to any moiety capable of beingdetected (e.g., primary labels and secondary labels). A “detectablemoiety” or “label” is the radical of a detectable compound.

“Primary” labels include radioisotope-containing moieties (e.g.,moieties that contain ³²P, ³³P, ³⁵S, or ¹⁴C), mass-tags, and fluorescentlabels, and are signal-generating reporter groups which can be detectedwithout further modifications.

Other primary labels include those useful for positron emissiontomography including molecules containing radioisotopes (e.g. ¹⁸F) orligands with bound radioactive metals (e.g. ⁶²Cu). In other embodiments,primary labels are contrast agents for magnetic resonance imaging suchas gadolinium, gadolinium chelates, or iron oxide (e.g Fe₃O₄ and Fe₂O₃)particles. Similarly, semiconducting nanoparticles (e.g. cadmiumselenide, cadmium sulfide, cadmium telluride) are useful as fluorescentlabels. Other metal nanoparticles (e.g colloidal gold) also serve asprimary labels.

“Secondary” labels include moieties such as biotin, or protein antigens,that require the presence of a second compound to produce a detectablesignal. For example, in the case of a biotin label, the second compoundmay include streptavidin-enzyme conjugates. In the case of an antigenlabel, the second compound may include an antibody-enzyme conjugate.Additionally, certain fluorescent groups can act as secondary labels bytransferring energy to another compound or group in a process ofnonradiative fluorescent resonance energy transfer (FRET), causing thesecond compound or group to then generate the signal that is detected.

Unless otherwise indicated, radioisotope-containing moieties areoptionally substituted hydrocarbon groups that contain at least oneradioisotope. Unless otherwise indicated, radioisotope-containingmoieties contain from 1-40 carbon atoms and one radioisotope. In certainembodiments, radioisotope-containing moieties contain from 1-20 carbonatoms and one radioisotope.

The term “mass-tag” as used herein refers to any compound that iscapable of being uniquely detected by virtue of its mass using massspectrometry (MS) detection techniques. Examples of mass-tags includeelectrophore release tags such asN-[3-[4′-[(p-methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]-isonipecoticacid, 4′-[2,3,5,6-tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags.

The terms “fluorescent label”, “fluorescent group”, “fluorescentcompound”, “fluorescent dye”, and “fluorophore”, as used herein, referto compounds or moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent compounds include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, anthracene, BODIPY dyes(BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650,BODIPY 650/665), carbazole, Carboxyrhodamine 6G, carboxy-X-rhodamine(ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy₃,Cy₅, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, and Texas Red-X.

The term “substrate”, as used herein refers to any material ormacromolecular complex to which a functionalized end-group of a PEG canbe attached. Examples of commonly used substrates include, but are notlimited to, glass surfaces, silica surfaces, plastic surfaces, metalsurfaces, surfaces containing a metallic or chemical coating, membranes(e.g., nylon, polysulfone, silica), micro-beads (e.g., latex,polystyrene, or other polymer), porous polymer matrices (e.g.,polyacrylamide gel, polysaccharide, polymethacrylate), andmacromolecular complexes (e.g., protein, polysaccharide).

The term “targeting group”, as used herein refers to any molecule,macromolecule, or biomacromolecule which selectively binds to receptorsthat are over-expressed on specific cell types. Such molecules can beattached to the functionalized end-group of a PEG for cell specificdelivery of proteins, viruses, DNA plasmids, oligonucleotides (e.g.siRNA, miRNA, antisense therapeutics, aptamers, etc.), drugs, dyes, andprimary or secondary labels which are bound to the opposite PEGend-group. Such targeting groups include, but or not limited tomonoclonal and polyclonal antibodies (e.g. IgG, IgA, IgM, IgD, IgEantibodies), sugars (e.g. mannose, mannose-6-phosphate, galactose),proteins (e.g. transferrin), oligopeptides (e.g. cyclic and acylicRGD-containing oligopeptides), oligonucleotides (e.g. aptamers), andvitamins (e.g. folate).

The term “permeation enhancer”, as used herein refers to any molecule,macromolecule, or biomacromolecule which aids in or promotes thepermeation of cellular membranes and/or the membranes of intracellularcompartments (e.g. endosome, lysosome, etc.) Such molecules can beattached to the functionalized end-group of a PEG to aid in theintracellular and/or cytoplasmic delivery of proteins, viruses, DNAplasmids, oligonucleotides (e.g. siRNA, miRNA, antisense therapeutics,aptamers, etc.), drugs, dyes, and primary or secondary labels which arebound to the opposite PEG end-group. Such permeation enhancers include,but are not limited to, oligopeptides containing protein transductiondomains such as the HIV-1Tat peptide sequence (GRKKRRQRRR),oligoarginine (RRRRRRRRR), or penetratin (RQIKIWFQNRRMKWKK).Oligopeptides which undergo conformational changes in varying pHenvironments such oligohistidine (HHHHH) also promote cell entry andendosomal escape.

3. Description of Exemplary Embodiments

As defined generally above, the n group of formula I is 10-2500. Incertain embodiments, the present invention provides compounds of formulaI, as described above, wherein n is about 225. In other embodiments, nis about 10 to about 40. In other embodiments, n is about 40 to about60. In other embodiments, n is about 60 to about 90. In still otherembodiments, n is about 90 to about 150. In other embodiments, n isabout 150 to about 200. In still other embodiments, n is about 200 toabout 250. In other embodiments, n is about 300 to about 375. In otherembodiments, n is about 400 to about 500. In still other embodiments, nis about 650 to about 750. In certain embodiments, n is selected from50±10. In other embodiments, n is selected from 80±10, 115±10, 180±10,or 225±10.

According to another embodiment, the present invention provides acompound of formula I, as described above, wherein said compound has apolydispersity index (“PDI”) of about 1.0 to about 1.2. According toanother embodiment, the present invention provides a compound of formulaI, as described above, wherein said compound has a polydispersity index(“PDI”) of about 1.02 to about 1.05. According to yet anotherembodiment, the present invention provides a compound of formula I, asdescribed above, wherein said compound has a polydispersity index(“PDI”) of about 1.05 to about 1.10. In other embodiments, said compoundhas a PDI of about 1.01 to about 1.03. In other embodiments, saidcompound has a PDI of about 1.10 to about 1.15. In still otherembodiments, said compound has a PDI of about 1.15 to about 1.20.

In certain embodiments, the present invention provides a compound offormula I, as described above, wherein the R¹ and R² groups of formula Iare different from each other.

In other embodiments, the present invention provides a compound offormula I, as described above, wherein only one of -L¹-R¹ and -L²-R² isa hydroxyl group.

In still other embodiments, the present invention provides a compound offormula I, as described above, wherein neither of -L¹-R¹ and -L²-R² is ahydroxyl group.

As defined generally above, R¹ is hydrogen, halogen, NO₂, CN, N₃,—N═C═O, —C(R)═NN(R)₂, —P(O)(OR)₂, —P(O)(X)₂, a 9-30-membered crownether, or an optionally substituted group selected from aliphatic, a 3-8membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, an8-10 membered saturated, partially unsaturated, or aryl bicyclic ringhaving 0-5 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a detectable moiety; wherein each R is independently hydrogenor an optionally substituted aliphatic group.

In certain embodiments, R¹ is optionally substituted aliphatic. In otherembodiments, R¹ is an unsubstituted aliphatic. In some embodiments, saidR¹ moiety is an optionally substituted alkyl group. In otherembodiments, said R¹ moiety is an optionally substituted alkynyl oralkenyl group. Such groups include t-butyl, 5-norbornene-2-yl,octane-5-yl, —C≡CH, —CH₂C≡CH, —CH₂CH₂C≡CH, and —CH₂CH₂CH₂C≡CH. When saidR¹ moiety is a substituted aliphatic group, suitable substituents on R¹include any of CN, N₃, NO₂, —CO₂H, —SH, —NH₂, —C(O)H, —NHC(O)R^(◯),—NHC(S)R^(◯), —NHC(O)NR^(◯) ₂, —NHC(S)NR^(◯) ₂, —NHC(O)OR^(◯),—NHNHC(O)R^(◯), —NHNHC(O)NR^(◯) ₂, —NHNHC(O)OR^(◯), —C(O)R^(◯),—C(S)R^(◯), —C(O)OR^(◯), —C(O)SR^(◯), —C(O)OSiR^(◯) ₃, —OC(O)R^(◯),SC(S)SR^(◯), —SC(O)R^(◯), —C(O)N(R^(◯)) ₂, —C(S)N(R^(◯)) ₂, —C(S)SR^(◯),—SC(S)SR^(◯)), —OC(O)N(R^(◯)) ₂, —C(O)NHN(R^(◯)) ₂, —C(O)N(OR^(◯)R^(◯),—C(O)C(O)R^(◯), —C(O)CH₂C(O)R^(◯), —C(NOR^(◯)R^(◯), —SSR^(◯),—S(O)₂R^(◯), —S(O)₂OR^(◯), —OS(O)₂R^(◯)), —S(O)₂N(R^(◯)) ₂,—S(O)R^(◯))₂, —N(R^(◯)S(O)₂N(R^(◯)) ₂, —N(R^(◯)S(O)₂R^(◯)),—N(OR^(◯)R^(◯)), —C(NH)N(R^(◯)) ₂, —P(O)₂R^(◯)), —P(O)(R^(◯)) ₂,—OP(O)(R^(◯)) ₂, or —OP(O)(OR^(◯))₂, wherein each R^(◯) is as definedherein.

In other embodiments, R¹ is an aliphatic group optionally substitutedwith any of Cl, Br, I, F, —NH2, —OH, —SH, —CO₂H, —C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH₂, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH—, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂. In other embodiments, the R¹ group offormula I is an optionally substituted aliphatic group havingsubstituents as depicted in the Appendix.

In certain embodiments, the R¹ group of formula I is a group suitablefor Click chemistry. Click reactions tend to involve high-energy(“spring-loaded”) reagents with well-defined reaction coordinates, thatgive rise to selective bond-forming events of wide scope. Examplesinclude nucleophilic trapping of strained-ring electrophiles (epoxide,aziridines, aziridinium ions, episulfonium ions), certain carbonylreactivity (e.g., the reaction between aldehydes and hydrazines orhydroxylamines), and several cycloaddition reactions. The azide-alkyne1,3-dipolar cycloaddition is one such reaction. Click chemistry is knownin the art and one of ordinary skill in the art would recognize thatcertain R¹ moieties of the present invention are suitable for Clickchemistry.

According to one embodiment, the R¹ group of formula I is anazide-containing group. According to another embodiment, the R¹ group offormula I is an alkyne-containing group. In certain embodiments, the R¹group of formula I has a terminal alkyne moiety. According to anotherembodiment, the R¹ group of formula I is an aldehyde-containing group.In certain embodiments, the R¹ group of formula I has a terminalhydrazine moiety. In other embodiments, the R¹ group of formula I has aterminal oxyamine moiety. In still other embodiments, the R¹ group offormula I is a epoxide-containing group. In certain other embodiments,the R¹ group of formula I has a terminal maleimide moiety.

In other embodiments, R¹ is an optionally substituted 3-8 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered saturated, partially unsaturated, or aryl bicyclic ring having0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In certain embodiments, R¹ is an optionally substituted 5-7 memberedsaturated or partially unsaturated ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted phenyl ring or a 5-6membered heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In certain embodiments, the R¹ group of formula I is an optionallysubstituted aryl group. Examples include optionally substituted phenyl,optionally substituted pyridyl, optionally substituted naphthyl,optionally substituted pyrenyl, optionally substituted triazole,optionally substituted imidazole, optionally substituted phthalimide,optionally substituted tetrazole, optionally substituted furan, andoptionally substituted pyran. When said R¹ moiety is a substituted arylgroup, suitable substituents on R¹ include any of R^(◯), CN, N₃, NO₂,—CH₃, —CH₂N₃, t-butyl, 5-norbornene-2-yl, octane-5-yl, —CH═CH₂, —C≡CH,—CH₂C≡CH, —CH₂CH₂C≡CH, —CH₂CH₂CH₂C≡CH, Cl, Br, I, F, —NH₂, —OH, —SH,—CO₂H, —C(O)H, —CH₂NH₂, —CH₂OH, —CH₂SH, —CH₂CO₂H, —CH₂C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH—, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH₂, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂.

Suitable substitutents on R¹ further includebis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embodiments, R¹ is hydrogen.

In certain embodiments, R¹ is N₃.

In other embodiments, R¹ is an epoxide ring.

According to certain embodiments, R¹ is methyl.

According to other embodiments, R¹ is —NH₂.

In certain embodiments, the R¹ group of formula I is a crown ether.Examples of such crown ethers include 12-crown-4,15-crown-5, and18-crown-6.

In still other embodiments, R¹ is a detectable moiety. Detectablemoieties are known in the art and include those described herein.According to one aspect of the invention, the R¹ group of formula I is afluorescent moiety. Such fluorescent moieties are well known in the artand include coumarins, quinolones, benzoisoquinolones, hostasol, andRhodamine dyes, to name but a few. Exemplary fluorescent moieties of R¹include anthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylateof rhodamine B, and the carboxylate of coumarin 343. In certainembodiments, R¹ is a detectable moiety selected from:

wherein each wavy line indicates the point of attachment to the rest ofthe molecule.

In certain embodiments, R¹ is —P(O)(OR)₂, or —P(O)(halogen)₂. Accordingto one aspect, the present invention provides a compound of formula I,wherein R¹ is —P(O)(OH)₂. According to another aspect, the presentinvention provides a compound of formula I, wherein R¹ is —P(O)(Cl)₂.

According to one embodiment, the R¹ group of formula I is selected fromany of those depicted in Tables 1 through 25.

As defined generally above, the L¹ group of formula I is a valence bondor a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂hydrocarbon chain, wherein 0-6 methylene units of L¹ are independentlyreplaced by -Cy-, —O—, —NR—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—,—NRSO₂—, —SO₂NR—, —NRC(O)—, —C(O)NR—, —OC(O)NR—, or —NRC(O)O—, whereineach -Cy- is independently an optionally substituted 3-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran optionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, L¹ is a valence bond. In other embodiments, L¹is a bivalent, saturated C₁₋₁₂ hydrocarbon chain, wherein 0-6 methyleneunits of L¹ are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—,—C(O)O—, —C(O)—, —C(O)NH—, or —NHC(O)—, wherein each -Cy- isindependently an optionally substituted 3-8 membered bivalent,saturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or anoptionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In still other embodiments,L¹ is a bivalent, saturated C₁₋₆ alkylene chain, wherein 0-3 methyleneunits of L¹ are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—,—C(O)O—, —C(O)—, —C(O)NH—, or —NHC(O)—.

In certain embodiments, L¹ is a C₁₋₆ alkylene chain wherein onemethylene unit of L′ is replaced by -Cy-. In other embodiments, L¹ is-Cy- (i.e. a C₁₋₆ alkylene chain wherein the methylene unit is replacedby -Cy-), wherein -Cy- is an optionally substituted 3-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.According to one aspect of the present invention, -Cy- is an optionallysubstituted bivalent aryl group. According to another aspect of thepresent invention, -Cy- is an optionally substituted bivalent phenylgroup. In other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated carbocyclic ring. In still otherembodiments, -Cy- is an optionally substituted 5-8 membered bivalent,saturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. Exemplary -Cy- groups includebivalent rings selected from phenyl, pyridyl, pyrimidinyl, cyclohexyl,cyclopentyl, or cyclopropyl.

In certain embodiments, the L¹ group of formula I is —O—, —S—, —NH—, or—C(O)O—. In other embodiments, the L¹ group of formula I is -Cy-,—C(O)—, —C(O)NH—, —NHC(O)—, —NH—O—, or —O-Cy-CH₂NH—O—. In still otherembodiments, the L¹ group of formula I is any of —OCH₂—, —OCH₂C(O)—,—OCH₂CH₂C(O)—, —OCH₂CH₂O—, —OCH₂CH₂S—, —OCH₂CH₂C(O)O—, —OCH₂CH₂NH—,—OCH₂CH₂NHC(O)—, —OCH₂CH₂C(O)NH—, and α—NHC(O)CH₂CH₂C(O)O—. According toanother aspect, the L¹ group of formula I is any of—OCH₂CH₂NHC(O)CH₂CH₂C(O)O—, —OCH₂CH₂NHC(O)CH₂OCH₂C(O) O—,—OCH₂CH₂NHC(O)CH₂OCH₂C(O)NH—, —CH₂C(O)NH—, —CH₂C(O)NHNH—, or—OCH₂CH₂NHNH—. In certain embodiments, L¹ is a C₁₋₆ alkylene chainwherein one methylene unit of L¹ is replaced by —O—. In otherembodiments, L¹ is —O—. Exemplary L¹ groups of formula I include any ofthose depicted in any of Tables 1 through 25.

According to another aspect of the present invention, a functional groupformed by the -L¹-R¹ moiety of formula I is optionally protected. Thus,in certain embodiments, the -L¹-R¹ moiety of formula I optionallycomprises a mono-protected amine, a di-protected amine, a protectedaldehyde, a protected hydroxyl, a protected carboxylic acid, or aprotected thiol group.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benzylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl ethers includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Alkoxyalkyl ethers include acetals such asmethoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethersinclude benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl,O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,p-cyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Suitable mono-protected amines furtherinclude, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected aminomoieties include t-butyloxycarbonylamino (-NHBOC),ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxycarbonylamino, allyloxycarbonylamino(-NHAlloc),benzyloxocarbonylamino (-NHCBZ), allylamino, benzylamino(-NHBn),fluorenylmethylcarbonyl(-NHFmoc), formamido, acetamido, chloroacetamido,dichloroacetamido, trifluoroacetamido, phenylacetamido,trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like.Suitable di-protected amines include amines that are substituted withtwo substituents independently selected from those described above asmono-protected amines, and further include cyclic imides, such asphthalimide, maleimide, succinimide, and the like. Suitable di-protectedamines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected aldehydesfurther include, but are not limited to, acyclic acetals, cyclicacetals, hydrazones, imines, and the like. Examples of such groupsinclude dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzylacetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes,semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected carboxylicacids further include, but are not limited to, optionally substitutedC₁₋₅ aliphatic esters, optionally substituted aryl esters, silyl esters,activated esters, amides, hydrazides, and the like. Examples of suchester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,benzyl, and phenyl ester, wherein each group is optionally substituted.Additional suitable protected carboxylic acids include oxazolines andortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Suitable protected thiols further include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, and thiocarbamates, and the like. Examplesof such groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

As defined generally above, the R² group of formula I is hydrogen,halogen, NO₂, CN, N₃, —N═C═O, —C(R)═NN(R)₂, —P(O)(OR)₂, —P(O)(X)₂, a9-30-membered crown ether, or an optionally substituted group selectedfrom aliphatic, a 3-8 membered saturated, partially unsaturated, or arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety, wherein each R isindependently hydrogen or an optionally substituted aliphatic group.

In certain embodiments, R² is optionally substituted aliphatic. In otherembodiments, R² is an unsubstituted aliphatic. In some embodiments, saidR² moiety is an optionally substituted alkyl group. In otherembodiments, said R² moiety is an optionally substituted alkynyl oralkenyl group. Such groups include t-butyl, 5-norbornene-2-yl,octane-5-yl, —CH₂C≡CH, —CH₂CH₂C≡CH, and —CH₂CH₂CH₂C≡CH. When said R²moiety is a substituted aliphatic group, suitable substituents on R²include any of CN, N₃, NO₂, —CO₂H, —SH, —NH₂, —C(O)H, —NHC(O)R^(◯),—NHC(S)R^(◯), —NHC(O)N(R^(◯)) ₂, —NHC(S)N(R^(◯)) ₂, —NHC(O)OR^(◯),—NHNHC(O)R^(◯)), —NHNHC(O)N(R^(◯)) ₂, —NHNHC(O)OR^(◯), —C(O)R^(◯),—C(S)R^(◯), —C(O)OR^(◯), —C(O)SR^(◯)), —C(O)OSi(R^(◯) ₃, —OC(O)R^(◯),SC(S)SR^(◯), —SC(O)R^(◯), —C(O)NR^(◯) ₂, —C(S)NR^(◯) ₂, —C(S)SR^(◯);—SC(S)SR^(◯)), —OC(O)N(R^(◯) ₂; —C(O)NHN(R^(◯)) ₂, —C(O)N(OR^(◯)R^(◯),—C(O)C(O)R^(◯), —C(O)CH₂C(O)R^(◯)), —C(NOR^(◯)R^(◯), —SSR^(◯),—S(O)₂R^(◯), —S(O)₂OR^(◯), —OS(O)₂R^(◯)), —S(O)₂N(R^(◯)) ₂, —S(O)R^(◯),—N(R^(◯)S(O)₂N(R^(◯)) ₂, —N(R^(◯)S(O)₂R^(◯)), —N(OR^(◯)R^(◯)),—C(NH)N(R^(◯)) ₂, —P(O)₂R^(◯), —P(O)(R^(◯)) ₂, —OP(O)(R^(◯)) ₂,or)-OP(O)(OR^(◯))₂, wherein each R^(◯) is as defined herein.

In other embodiments, R² is an aliphatic group optionally substitutedwith any of Cl, Br, I, F, —NH₂, —OH, —SH, —CO₂H, —C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH—, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH₂, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂. In other embodiments, the R² group offormula I is an optionally substituted aliphatic group havingsubstituents as depicted in any of Tables 1 through 25.

In certain embodiments, the R² group of formula I is a group suitablefor Click chemistry. Click reactions tend to involve high-energy(“spring-loaded”) reagents with well-defined reaction coordinates, thatgive rise to selective bond-forming events of wide scope. Examplesinclude nucleophilic trapping of strained-ring electrophiles (epoxide,aziridines, aziridinium ions, episulfonium ions), certain carbonylreactivity (e.g., the reaction between aldehydes and hydrazines orhydroxylamines), and several cycloaddition reactions. The azide-alkyne1,3-dipolar cycloaddition is one such reaction. Click chemistry is knownin the art and one of ordinary skill in the art would recognize thatcertain R² moieties of the present invention are suitable for Clickchemistry.

According to one embodiment, the R² group of formula I is anazide-containing group. According to another embodiment, the R² group offormula I is an alkyne-containing group. In certain embodiments, the R²group of formula I has a terminal alkyne moiety. According to anotherembodiment, the R² group of formula I is an aldehyde-containing group.In certain embodiments, the R² group of formula I has a terminalhydrazine moiety. In other embodiments, the R² group of formula I has aterminal oxyamine moiety. In still other embodiments, the R² group offormula I is a epoxide-containing group. In certain other embodiments,the R² group of formula I has a terminal maleimide moiety.

In other embodiments, R² is an optionally substituted 3-8 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered saturated, partially unsaturated, or aryl bicyclic ring having0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In certain embodiments, R² is an optionally substituted 3-7 memberedsaturated or partially unsaturated ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R² is an optionally substituted phenyl ring or a 5-6membered heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In certain embodiments, the R² group of formula I is an optionallysubstituted aryl group. Examples include optionally substituted phenyl,optionally substituted pyridyl, optionally substituted naphthyl,optionally substituted pyrenyl, optionally substituted triazole,optionally substituted imidazole, optionally substituted phthalimide,optionally substituted tetrazole, optionally substituted furan, andoptionally substituted pyran. When said R² moiety is a substituted arylgroup, suitable substituents on R² include R^(◯), CN, N₃, NO₂, —CH₃,—CH₂N₃, t-butyl, 5-norbornene-2-yl, octane-5-yl, —CH═CH₂, —CH₂C≡CH,—CH₂CH₂C≡CH, —CH₂CH₂CH₂C≡CH, Cl, Br, I, F, —NH₂, —OH, —SH, —CO₂H,—C(O)H, —CH₂NH₂, —CH₂OH, —CH₂SH, —CH₂CO₂H, —CH₂C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH—, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH—, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O) (C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂.

Suitable substitutents on R² further includebis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embodiments, R² is hydrogen.

In certain embodiments, R² is N₃.

In other embodiments, R² is an epoxide ring.

In certain embodiments, R² is Me. In other embodiments, R² is —NH₂

In certain embodiments, the R² group of formula I is a crown ether.Examples of such crown ethers include 12-crown-4,15-crown-5, and18-crown-6.

In still other embodiments, R² is a detectable moiety. Detectablemoieties are known in the art and include those described herein.According to one aspect of the invention, the R² group of formula I is afluorescent moiety. Such fluorescent moieties are well known in the artand include coumarins, quinolones, benzoisoquinolones, hostasol, andRhodamine dyes, to name but a few. Exemplary fluorescent moieties of R²include anthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, the carboxylateof rhodamine B, and the carboxylate of coumarin 343. In certainembodiments, R² is a detectable moiety selected from:

wherein each wavy line indicates the point of attachment to the rest ofthe molecule.

In certain embodiments, R² is —P(O)(OR)₂, or —P(O)(X)₂. According to oneaspect, the present invention provides a compound of formula I, whereinR² is —P(O)(OH)₂. According to another aspect, the present inventionprovides a compound of formula I, wherein R² is —P(O)(Cl)₂.

In certain embodiments, the R² group of formula I is selected from anyof those depicted in any of Tables 1 through 25

As defined generally above, the L² group of formula I is a valence bondor a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂hydrocarbon chain, wherein 0-6 methylene units of L² are independentlyreplaced by -Cy-, —O—, —NR—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—,—NRSO₂—, —SO₂NR—, —NRC(O)—, —C(O)NR—, —OC(O)NR—, —NRC(O)O—, —NH—O—, or—O-Cy-CH₂NH—O—, wherein each -Cy- is independently an optionallysubstituted 3-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or an optionally substituted 8-10 membered bivalentsaturated, partially unsaturated, or aryl bicyclic ring having 0-5heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, L² is a valence bond. In other embodiments, L²is a bivalent, saturated C₁₋₁₂ alkylene chain, wherein 0-6 methyleneunits of L² are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—,—C(O)O—, —C(O)—, —C(O)NH—, or —NHC(O)—, wherein each -Cy- isindependently an optionally substituted 5-8 membered bivalent,saturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or anoptionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In still other embodiments,L² is a bivalent, saturated C₁₋₆ alkylene chain, wherein 0-3 methyleneunits of L² are independently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—,—C(O)O—, —C(O)—, —C(O)NH—, or —NHC(O)—.

In certain embodiments, L² is a C₁₋₆ alkylene chain wherein onemethylene unit of L² is replaced by -Cy- or —OCy-. In other embodiments,L² is -Cy- (i.e. a C₁₋₆ alkylene chain wherein the methylene unit isreplaced by -Cy-), wherein -Cy- is an optionally substituted 3-8membered bivalent, saturated, partially unsaturated, or aryl ring having0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.According to one aspect of the present invention, -Cy- is an optionallysubstituted bivalent aryl group. According to another aspect of thepresent invention, -Cy- is an optionally substituted bivalent phenylgroup. In other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated carbocyclic ring. In still otherembodiments, -Cy- is an optionally substituted 5-8 membered bivalent,saturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. Exemplary -Cy- groups includebivalent rings selected from phenyl, pyridyl, pyrimidinyl, cyclohexyl,cyclopentyl, or cyclopropyl.

In certain embodiments, L² is —O-Cy- (i.e. a C₂ alkylene chain whereinone methylene unit is replaced by -Cy- and the other by —O—), wherein-Cy- is an optionally substituted 3-8 membered bivalent, saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. According to one aspect ofthe present invention, -Cy- is an optionally substituted bivalent arylgroup. According to another aspect of the present invention, -Cy- is anoptionally substituted bivalent phenyl group. In other embodiments, -Cy-is an optionally substituted 5-8 membered bivalent, saturatedcarbocyclic ring. In still other embodiments, -Cy- is an optionallysubstituted 5-8 membered bivalent, saturated heterocyclic ring having1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.Exemplary -Cy- groups include bivalent rings selected from phenyl,pyridyl, pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

In certain embodiments, the L² group of formula I is —O—, —S—, —NH—, or—C(O)O—. In other embodiments, the L² group of formula I is -Cy-,—C(O)—, —C(O)NH—, —NH—O—, —O-Cy-CH₂NH—O—, or —NHC(O)—. In still otherembodiments, the L² group of formula I is any of —OCH₂—, —OCH₂C(O)—,—OCH₂CH₂C(O)—, —OCH₂CH₂O—, —OCH₂CH₂S—, —OCH₂CH₂C(O)O—, —OCH₂CH₂NH—,—OCH₂CH₂NHC(O)—, —OCH₂CH₂C(O)NH—, and —NHC(O)CH₂CH₂C(O)O—. According toanother aspect, the L² group of formula I is any of—OCH₂CH₂NHC(O)CH₂CH₂C(O)O—, —OCH₂CH₂NHC(O)CH₂OCH₂C(O)O—,—OCH₂CH₂NHC(O)CH₂OCH₂C(O)NH—, —CH₂C(O)NH—, —CH₂C(O)NHNH—, or—OCH₂CH₂NHNH—. In other embodiments, the L² group of formula I is—OC(O)CH₂CH₂CH₂CH₂—, —OCH₂CH₂—, —NHC(O)CH₂CH₂—, —NHC(O)CH₂CH₂CH₂—,—OC(O)CH₂CH₂CH₂—, —O-Cy-, —O-Cy-CH₂—, —O-Cy-NH—, —O-Cy-S—, —O-Cy-C(O)—,—O-Cy-C(O)O—, —O-Cy-C(O)O-Cy-, —O-Cy-OCH₂CH(CH₃)C(O)O—, —O-Cy-C(O)O—,—O-Cy-OCH(CH₃)CH₂C(O)O—, —OCH₂C(O)O—, —OCH₂C(O)NH—, —OCH₂O—, —OCH₂S—, or—OCH₂NH—. In certain embodiments, L² is —O—. Exemplary L² groups offormula I include any of those depicted in any of Tables 1 through 25.

According to another aspect of the present invention, a functional groupformed by the -L²-R² moiety of formula I is optionally protected. Thus,in certain embodiments, the -L²-R² moiety of formula I optionallycomprises a mono-protected amine, a di-protected amine, a protectedaldehyde, a protected hydroxyl, a protected carboxylic acid, or aprotected thiol group. Such groups include those described above withrespect to the -L¹-R¹ moiety of formula I.

Exemplary compounds of formula I are set forth in the Appendix, whereineach n is as defined herein. In certain embodiments, the presentinvention provides any compound as depicted in the Appendix.

According to another aspect of the present invention, the R² group offormula I is —P(O)(OR)₂. Accordingly, the present invention provides acompound of formula IIa:

or a salt thereof, wherein:

-   m is 10-2500;-   R^(x) is hydrogen, halogen, NO₂, CN, N₃, —N═C═O, —C(R)═NN(R)₂,    —P(O)(OR)₂, —P(O)(X)₂, a 9-30 membered crown ether, or an optionally    substituted group selected from aliphatic, a 3-8 membered saturated,    partially unsaturated, or aryl ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, an 8-10    membered saturated, partially unsaturated, or aryl bicyclic ring    having 0-5 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or a detectable moiety;-   each X is independently halogen;-   each R is independently hydrogen or an optionally substituted group    selected from aliphatic or a a 3-8 membered saturated, partially    unsaturated, or aryl ring having 0-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   L^(x) is a valence bond or a bivalent, saturated or unsaturated,    straight or branched C₁₋₁₂ hydrocarbon chain, wherein 0-6 methylene    units of L^(x) are independently replaced by -Cy-, —O—, —NR—, —S—,    —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NRSO₂—, —SO₂NR—, —NRC(O)—,    —C(O)NR—, —OC(O)NR—, or —NRC(O)O—, wherein:    -   each -Cy- is independently an optionally substituted 3-8        membered bivalent, saturated, partially unsaturated, or aryl        ring having 0-4 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, or an optionally substituted 8-10        membered bivalent saturated, partially unsaturated, or aryl        bicyclic ring having 0-5 heteroatoms independently selected from        nitrogen, oxygen, or sulfur.

As defined generally above, the m group of formula IIa is 10-2500. Incertain embodiments, the present invention provides compounds of formulaIIa, as described above, wherein m is about 225. In other embodiments, mis about 10 to about 40. In other embodiments, m is about 40 to about60. In other embodiments, m is about 60 to about 90. In still otherembodiments, m is about 90 to about 150. In other embodiments, m isabout 150 to about 200. In still other embodiments, m is about 200 toabout 250. In other embodiments, m is about 300 to about 375. In otherembodiments, m is about 400 to about 500. In still other embodiments, mis about 650 to about 750.

According to another embodiment, the present invention provides acompound of formula IIa, as described above, wherein said compound has apolydispersity index (“PDI”) of about 1.0 to about 1.2. According toanother embodiment, the present invention provides a compound of formulaIIa, as described above, wherein said compound has a polydispersityindex (“PDI”) of about 1.02 to about 1.05. According to yet anotherembodiment, the present invention provides a compound of formula IIa, asdescribed above, wherein said compound has a polydispersity index(“PDI”) of about 1.05 to about 1.10. In other embodiments, said compoundhas a PDI of about 1.01 to about 1.03. In other embodiments, saidcompound has a PDI of about 1.10 to about 1.15. In still otherembodiments, said compound has a PDI of about 1.15 to about 1.20.

In other embodiments, the present invention provides a compound offormula IIa, as described above, wherein -L^(x)-R^(x) is a hydroxylgroup.

As defined generally above, the R^(x) group of formula IIa is hydrogen,halogen, NO₂, CN, N₃, —N═C═O, —C(R)═NN(R)₂, —P(O)(OR)₂, —P(O)(halogen)₂,a 9-30-membered crown ether, or an optionally substituted group selectedfrom aliphatic, a 3-8 membered saturated, partially unsaturated, or arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety; wherein each R isindependently hydrogen or an optionally substituted aliphatic group.

In certain embodiments, the R^(x) group of formula IIa is optionallysubstituted aliphatic. In other embodiments, R^(x) is an unsubstitutedaliphatic. In some embodiments, said R^(x) moiety is an optionallysubstituted alkyl group. In other embodiments, said R^(x) moiety is anoptionally substituted alkynyl or alkenyl group. Such groups includet-butyl, 5-norbornene-2-yl, octane-5-yl, -0=CH, —CH₂C≡CH, —CH₂CH₂C≡CH,and —CH₂CH₂CH₂C≡CH. When said R^(x) moiety is a substituted aliphaticgroup, suitable substituents on R^(x) include any of CN, N₃, NO₂, —CO₂H,—SH, —NH₂, —C(O)H, —NHC(O)R^(◯), —NHC(S)R^(◯), —NHC(O)NR^(◯) ₂,—NHC(S)NR^(◯) ₂, —NHC(O)OR^(◯), —NHNHC(O)R^(◯), —NHNHC(O)NR^(◯) ₂,—NHNHC(O)OR^(◯), —C(O)R^(◯), —C(S)R^(◯), —C(O)OR^(◯), —C(O)SR^(◯),—C(O)OSiR^(◯) ₃, —OC(O)R^(◯), SC(S)SR^(◯), —SC(O)R^(◯)), —C(O)N(R^(◯))₂, —C(S)N(R^(◯)) ₂, —C(S)SR^(◯), —SC(S)SR^(◯)), —OC(O)N(R^(◯)) ₂,—C(O)NHN(R^(◯)) ₂, —C(O)N(OR^(◯)R^(◯), —C(O)C(O)R^(◯),—C(O)CH₂C(O)R^(◯)), —C(NOR^(◯)R^(◯), —SSR^(◯), —S(O)₂R^(◯),—S(O)₂OR^(◯), —OS(O)₂R^(◯), —S(O)₂N(R^(◯)) ₂, —S(O)R^(◯),—N(R^(◯)S(O)₂N(R^(◯)) ₂, —N(R^(◯)S(O)₂R^(◯)), —N(OR^(◯)R^(◯)),—C(NH)N(R^(◯)) ₂, —P(O)₂R^(◯)), —P(O)(R^(◯)) ₂, —OP(O)(R^(◯)) ₂, or—OP(O)(OR^(◯) ₂, wherein each R^(◯) is as defined herein.

In other embodiments, R^(x) is an aliphatic group optionally substitutedwith any of Cl, Br, I, F, —NH₂, —OH, —SH, —CO₂H, —C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH₂, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH—, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂. In other embodiments, the R^(x) group offormula IIa is an optionally substituted aliphatic group havingsubstituents as depictedin any of Tables 1 through 25.

In certain embodiments, the R^(x) group of formula IIa is a groupsuitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, that give rise to selective bond-forming events of widescope. Examples include nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain carbonyl reactivity (e.g., the reaction between aldehydesand hydrazines or hydroxylamines), and several cycloaddition reactions.The azide-alkyne 1,3-dipolar cycloaddition is one such reaction. Clickchemistry is known in the art and one of ordinary skill in the art wouldrecognize that certain R^(x) moieties of the present invention aresuitable for Click chemistry.

According to one embodiment, the R^(x) group of formula IIa is anazide-containing group. According to another embodiment, the R^(x) groupof formula IIa is an alkyne-containing group. In certain embodiments,the R^(x) group of formula IIa has a terminal alkyne moiety. Accordingto another embodiment, the R^(x) group of formula IIa is analdehyde-containing group. In certain embodiments, the R^(x) group offormula I has a terminal hydrazine moiety. In other embodiments, theR^(x) group of formula IIa has a terminal oxyamine moiety. In stillother embodiments, the R^(x) group of formula IIa is aepoxide-containing group. In certain other embodiments, the R^(x) groupof formula IIa has a terminal maleimide moiety.

In other embodiments, R^(x) is an optionally substituted 3-8 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered saturated, partially unsaturated, or aryl bicyclic ring having0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In certain embodiments, R^(x) is an optionally substituted 5-7 memberedsaturated or partially unsaturated ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R^(x) is an optionally substituted phenyl ring or a 5-6membered heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In certain embodiments, the R^(x) group of formula IIa is an optionallysubstituted aryl group. Examples include optionally substituted phenyl,optionally substituted pyridyl, optionally substituted naphthyl,optionally substituted pyrenyl, optionally substituted triazole,optionally substituted imidazole, optionally substituted phthalimide,optionally substituted tetrazole, optionally substituted furan, andoptionally substituted pyran. When said R^(x) moiety is a substitutedaryl group, suitable substituents on R¹ include R^(◯), CN, N₃, NO₂,—CH₃, —CH₂N₃, t-butyl, 5-norbornene-2-yl, octane-5-yl, —CH═CH₂, —C≡CH,—CH₂C≡CH, —CH₂CH₂C≡CH, —CH₂CH₂CH₂C≡CH, Cl, Br, I, F, —NH₂, —OH, —SH,—CO₂H, —C(O)H, —CH₂NH₂, —CH₂OH, —CH₂SH, —CH₂CO₂H, —CH₂C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH—, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH₂, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂.

Suitable substitutents on R^(x) further includebis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embodiments, R^(x) is hydrogen.

In certain embodiments, R^(x) is N₃.

In other embodiments, R^(x) is an epoxide ring.

In certain embodiments, R^(x) is methyl. In other embodiments, R^(x) is—NH₂.

In certain embodiments, the R^(x) group of formula IIa is a crown ether.Examples of such crown ethers include 12-crown-4,15-crown-5, and18-crown-6.

In still other embodiments, R^(x) is a detectable moiety. Detectablemoieties are known in the art and include those described herein.According to one aspect of the invention, the Rx group of formula IIa isa fluorescent moiety. Such fluorescent moieties are well known in theart and include coumarins, quinolones, benzoisoquinolones, hostasol, andRhodamine dyes, to name but a few. Exemplary fluorescent moieties ofR^(x) include anthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, thecarboxylate of rhodamine B, and the carboxylate of coumarin 343. Incertain embodiments, R^(x) is a detectable moiety selected from:

wherein each heavy line indicates the point of attachment to the rest ofthe molecule.

In certain embodiments, R^(x) is —P(O)(OR)₂, or —P(O)(X)₂. According toone aspect, the present invention provides a compound of formula IIa,wherein R^(x) is —P(O)(OH)₂. According to another aspect, the presentinvention provides a compound of formula IIa, wherein R^(x) is—P(O)(Cl)₂.

As defined generally above, the L^(x) group of formula IIa is a valencebond or a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂hydrocarbon chain, wherein 0-6 methylene units of L^(x) areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or—NHC(O)O—, wherein -Cy- is an optionally substituted 5-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran optionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, the L^(x) group of formula IIa is a valencebond. In other embodiments, L^(x) a bivalent, saturated C₁₋₁₂hydrocarbon chain, wherein 0-6 methylene units of L^(x) areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —C(O)NH—, or —NHC(O)—, wherein -Cy- is an optionally substituted3-8 membered bivalent, saturated, partially unsaturated, or aryl ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an optionally substituted 8-10 membered bivalent saturated,partially unsaturated, or aryl bicyclic ring having 0-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In still otherembodiments, L^(x) a bivalent, saturated C₁₋₆ alkylene chain, wherein0-3 methylene units of L^(x) are independently replaced by -Cy-, —O—,—NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —C(O)NH—, or —NHC(O)—,

In certain embodiments, L″ is -Cy- (i.e. a C₁₋₆ alkylene chain whereinthe methylene unit is replaced by -Cy-), wherein -Cy- is an optionallysubstituted 3-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to one aspect of the present invention,-Cy- is an optionally substituted bivalent aryl group. According toanother aspect of the present invention, -Cy- is an optionallysubstituted bivalent phenyl group. In other embodiments, -Cy- is anoptionally substituted 5-8 membered bivalent, saturated carbocyclicring. In still other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy-groups include bivalent rings selected from phenyl, pyridyl,pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

In certain embodiments, the L^(x) group of formula IIa is —O—, —S—,—NH—, or —C(O)O—. In other embodiments, the L^(x) group of formula IIais -Cy-, —C(O)—, —C(O)NH—, —NHC(O)—, —NH—O—, or —O-Cy-CH₂NH—O—. In stillother embodiments, the L^(x) group of formula IIa is any of —OCH₂—,—OCH₂C(O)—, —OCH₂CH₂C(O)—, —OCH₂CH₂O—, —OCH₂CH₂S—, —OCH₂CH₂C(O)O—,—OCH₂CH₂NH—, —OCH₂CH₂NHC(O)—, —OCH₂CH₂C(O)NH—, and —NHC(O)CH₂CH₂C(O)O—.According to another aspect, the L^(x) group of formula IIa is any of—OCH₂CH₂NHC(O)CH₂CH₂C(O)O—, —OCH₂CH₂NHC(O)CH₂OCH₂C(O)O—,—OCH₂CH₂NHC(O)CH₂OCH₂C(O)NH—, —CH₂C(O)NH—, —CH₂C(O)NHNH—, or—OCH₂CH₂NHNH—. Exemplary L^(x) groups of formula IIa include any ofthose depicted in any of Tables 1 through 25.

According to another aspect of the present invention, a functional groupformed by the -L^(x)-R^(x) moiety of formula IIa is optionallyprotected. Thus, in certain embodiments, the -L^(x)-R^(x) moiety offormula IIa optionally comprises a mono-protected amine, a di-protectedamine, a protected aldehyde, a protected hydroxyl, a protectedcarboxylic acid, or a protected thiol group. Such groups include thosedescribed above with respect to the -L¹-R¹ moiety of formula I.

According to yet another aspect of the present invention, the R² groupof formula I is —P(O)(X)₂. Accordingly, the present invention provides acompound of formula IIb:

or a salt thereof, wherein:

-   m is 10-2500;-   each X is independently halogen;-   R^(x) is hydrogen, halogen, NO₂, CN, N₃, —N═C—O, —C(R)═NN(R)₂,    —P(O)(OR)₂, —P(O)(X)₂, a 9-30 membered crown ether, or an optionally    substituted group selected from aliphatic, a 3-8 membered saturated,    partially unsaturated, or aryl ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur, an 8-10    membered saturated, partially unsaturated, or aryl bicyclic ring    having 0-5 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or a detectable moiety;-   each R is independently hydrogen or an optionally substituted group    selected from aliphatic or a a 3-8 membered saturated, partially    unsaturated, or aryl ring having 0-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur;-   L^(x) is a valence bond or a bivalent, saturated or unsaturated,    straight or branched C₁₋₁₂ hydrocarbon chain, wherein 0-6 methylene    units of L^(x) are independently replaced by -Cy-, —O—, —NR—, —S—,    —OC(O)—, —C(O)O—, —C(O)—, —SO—, —SO₂—, —NRSO₂—, —SO₂NR—, —NRC(O)—,    —C(O)NR—, —OC(O)NR—, or —NRC(O)O—, wherein:    -   each -Cy- is independently an optionally substituted 3-8        membered bivalent, saturated, partially unsaturated, or aryl        ring having 0-4 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, or an optionally substituted 8-10        membered bivalent saturated, partially unsaturated, or aryl        bicyclic ring having 0-5 heteroatoms independently selected from        nitrogen, oxygen, or sulfur.

As defined generally above, the m group of formula IIb is 10-2500. Incertain embodiments, the present invention provides compounds of formulaIIb, as described above, wherein m is about 225. In other embodiments, mis about 10 to about 40. In other embodiments, m is about 40 to about60. In other embodiments, m is about 60 to about 90. In still otherembodiments, m is about 90 to about 150. In other embodiments, m isabout 150 to about 200. In still other embodiments, m is about 200 toabout 250. In other embodiments, m is about 300 to about 375. In otherembodiments, m is about 400 to about 500. In still other embodiments, mis about 650 to about 750.

According to another embodiment, the present invention provides acompound of formula IIb, as described above, wherein said compound has apolydispersity index (“PDI”) of about 1.0 to about 1.2. According toanother embodiment, the present invention provides a compound of formulaIIb, as described above, wherein said compound has a polydispersityindex (“PDI”) of about 1.02 to about 1.05. According to yet anotherembodiment, the present invention provides a compound of formula IIb, asdescribed above, wherein said compound has a polydispersity index(“PDI”) of about 1.05 to about 1.10. In other embodiments, said compoundhas a PDI of about 1.01 to about 1.03. In other embodiments, saidcompound has a PDI of about 1.10 to about 1.15. In still otherembodiments, said compound has a PDI of about 1.15 to about 1.20.

In other embodiments, the present invention provides a compound offormula IIb, as described above, wherein -L^(x)-R^(x) is a hydroxylgroup.

As defined generally above, the R^(x) of formula IIb is hydrogen,halogen, NO₂, CN, N₃, —N═C═O, —C(R)═NN(R)₂, —P(O)(OR)₂, —P(O)(X)₂, a9-30-membered crown ether, or an optionally substituted group selectedfrom aliphatic, a 3-8 membered saturated, partially unsaturated, or arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, an 8-10 membered saturated, partially unsaturated, oraryl bicyclic ring having 0-5 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or a detectable moiety; wherein each R isindependently hydrogen or an optionally substituted aliphatic group.

In certain embodiments, the R^(x) group of formula IIb is optionallysubstituted aliphatic. In other embodiments, R^(x) is an unsubstitutedaliphatic. In some embodiments, said R^(x) moiety is an optionallysubstituted alkyl group. In other embodiments, said R^(x) moiety is anoptionally substituted alkynyl or alkenyl group. Such groups includet-butyl, 5-norbornene-2-yl, octane-5-yl, —C≡CH, —CH₂C≡CH, —CH₂CH₂C≡CH,and —CH₂CH₂CH₂C≡CH. When said R^(x) moiety is a substituted aliphaticgroup, suitable substituents on R^(x) include any of CN, N₃, NO₂, —CO₂H,—SH, —NH₂, —C(O)H, —NHC(O)R^(◯), —NHC(S)R^(◯), —NHC(O)NR^(◯) ₂,—NHC(S)NR^(◯) ₂, —NHC(O)OR^(◯), —NHNHC(O)R^(◯), —NHNHC(O)NR^(◯) ₂,—NHNHC(O)OR^(◯), —C(O)R^(◯), —C(S)R^(◯), —C(O)OR^(◯), —C(O)SR^(◯),—C(O)OSiR^(◯) ₃, —OC(O)R^(◯), SC(S)SR^(◯), —SC(O)R^(◯)), —C(O)N(R^(◯))₂, —C(S)N(R^(◯)) ₂, —C(S)SR^(◯), —SC(S)SR^(◯)), —OC(O)N(R^(◯)) ₂,—C(O)NHN(R^(◯)) ₂, —C(O)N(OR^(◯)R^(◯), —C(O)C(O)R^(◯),—C(O)CH₂C(O)R^(◯)), —C(NOR^(◯)R^(◯), —SSR^(◯), —S(O)₂R^(◯),—S(O)₂OR^(◯), —OS(O)₂R^(◯), —S(O)₂N(R^(◯)) ₂, —S(O)R^(◯),—N(R^(◯)S(O)₂N(R^(◯)) ₂, —N(R^(◯)S(O)₂R^(◯)), —N(OR^(◯)R^(◯),—C(NH)N(R^(◯))₂, —P(O)₂R^(◯)), —P(O)(R^(◯)) ₂, —OP(O)(R^(◯)) ₂, or—OP(O)(OR^(◯) ₂, wherein each R^(◯) is as defined herein.

In other embodiments, R^(x) is an aliphatic group optionally substitutedwith any of Cl, Br, I, F, —NH₂, —OH, —SH, —CO₂H, —C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH₂, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH—, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C₁₋₆ aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂. In other embodiments, the R^(x) group offormula IIb is an optionally substituted aliphatic group havingsubstituents as depicted for R¹ in any of Tables 1 through 25.

In certain embodiments, the R^(x) group of formula IIb is a groupsuitable for Click chemistry. Click reactions tend to involvehigh-energy (“spring-loaded”) reagents with well-defined reactioncoordinates, that give rise to selective bond-forming events of widescope. Examples include nucleophilic trapping of strained-ringelectrophiles (epoxide, aziridines, aziridinium ions, episulfoniumions), certain carbonyl reactivity (e.g., the reaction between aldehydesand hydrazines or hydroxylamines), and several cycloaddition reactions.The azide-alkyne 1,3-dipolar cycloaddition is one such reaction. Clickchemistry is known in the art and one of ordinary skill in the art wouldrecognize that certain R^(x) moieties of the present invention aresuitable for Click chemistry.

According to one embodiment, the R^(x) group of formula IIb is anazide-containing group. According to another embodiment, the R^(x) groupof formula IIb is an alkyne-containing group. In certain embodiments,the R^(x) group of formula IIb has a terminal alkyne moiety. Accordingto another embodiment, the R^(x) group of formula IIb is analdehyde-containing group. In certain embodiments, the R^(x) group offormula IIb has a terminal hydrazine moiety. In other embodiments, theR^(x) group of formula IIb has a terminal oxyamine moiety. In stillother embodiments, the R^(x) group of formula IIb is aepoxide-containing group. In certain other embodiments, the R^(X) groupof formula IIb has a terminal maleimide moiety.

In other embodiments, R^(x) is an optionally substituted 5-8 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, an 8-10membered saturated, partially unsaturated, or aryl bicyclic ring having0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.In certain embodiments, R^(x) is an optionally substituted 5-7 memberedsaturated or partially unsaturated ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R^(x) is an optionally substituted phenyl ring or a 5-6membered heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In certain embodiments, the R^(x) group of formula IIb is an optionallysubstituted aryl group. Examples include optionally substituted phenyl,optionally substituted pyridyl, optionally substituted naphthyl,optionally substituted pyrenyl, optionally substituted triazole,optionally substituted imidazole, optionally substituted phthalimide,optionally substituted tetrazole, optionally substituted furan, andoptionally substituted pyran. When said R^(x) moiety is a substitutedaryl group, suitable substituents on R^(x) include R^(◯), CN, N₃, NO₂,—CH₃, —CH₂N₃, t-butyl, 5-norbornene-2-yl, octane-5-yl, —CH═CH₂, —C≡CH,—CH₂C≡CH, —CH₂CH₂C≡CH, —CH₂CH₂CH₂C≡CH, Cl, Br, I, F, —NH₂, —OH, —SH,—CO₂H, —C(O)H, —CH₂NH₂, —CH₂OH, —CH₂SH, —CH₂CO₂H, —CH₂C(O)H, —C(O)(C₁₋₆aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)NH—, —NHC(O)NH(C₁₋₆aliphatic), —NHC(S)NH₂, —NHC(S)N(C₁₋₆ aliphatic)₂, —NHC(O)O(C₁₋₆aliphatic), —NHNH₂, —NHNHC(O)(C₁₋₆ aliphatic), —NHNHC(O)NH₂,—NHNHC(O)NH(C₁₋₆ aliphatic), —NHNHC(O)O(C₁₋₆ aliphatic), —C(O)NH₂,—C(O)NH(C₁₋₆ aliphatic)₂, —C(O)NHNH₂, —C(S)N(C_(—6) aliphatic)₂,—OC(O)NH(C₁₋₆ aliphatic), —C(O)C(O)(C₁₋₆ aliphatic), —C(O)CH₂C(O)(C₁₋₆aliphatic), —S(O)₂(C₁₋₆ aliphatic), —S(O)₂O(C₁₋₆ aliphatic),—OS(O)₂(C₁₋₆ aliphatic), —S(O)₂NH(C₁₋₆ aliphatic), —S(O)(C₁₋₆aliphatic), —NHS(O)₂NH(C₁₋₆ aliphatic), —NHS(O)₂(C₁₋₆ aliphatic),—P(O)₂(C₁₋₆ aliphatic), —P(O)(C₁₋₆ aliphatic)₂, —OP(O)(C₁₋₆ aliphatic)₂,or —OP(O)(OC₁₋₆ aliphatic)₂.

Suitable substitutents on R^(x) further includebis-(4-ethynyl-benzyl)-amino, dipropargylamino, di-hex-5-ynyl-amino,di-pent-4-ynyl-amino, di-but-3-ynyl-amino, propargyloxy, hex-5-ynyloxy,pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxy-ethyldisulfanyl,2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl,2-propargyloxy-ethyldisulfanyl, bis-benzyloxy-methyl,[1,3]dioxolan-2-yl, and [1,3]dioxan-2-yl.

In other embodiments, R^(x) is hydrogen.

In certain embodiments, R^(x) is N₃.

In certain embodiments, R^(x) is an epoxide ring.

In certain embodiments, R^(x) is methyl. In other embodiments, R^(x) is—NH₂.

In certain embodiments, the R^(x) group of formula IIb is a crown ether.Examples of such crown ethers include 12-crown-4,15-crown-5, and18-crown-6.

In still other embodiments, R^(x) is a detectable moiety. Detectablemoieties are known in the art and include those described herein.According to one aspect of the invention, the R^(x) group of formula IIbis a fluorescent moiety. Such fluorescent moieties are well known in theart and include coumarins, quinolones, benzoisoquinolones, hostasol, andRhodamine dyes, to name but a few. Exemplary fluorescent moieties ofR^(x) include anthracen-9-yl, pyren-4-yl, 9-H-carbazol-9-yl, thecarboxylate of rhodamine B, and the carboxylate of coumarin 343. Incertain embodiments, R^(x) is a detectable moiety selected from:

wherein each wavy line indicates the point of attachment to the rest ofthe molecule.

In certain embodiments, R^(x) is —P(O)(OR)₂, or —P(O)(X)₂. According toone aspect, the present invention provides a compound of formula IIb,wherein R^(x) is —P(O)(OH)₂. According to another aspect, the presentinvention provides a compound of formula IIb, wherein R^(x) is—P(O)(Cl)₂.

As defined generally above, the L^(x) group of formula IIb is a valencebond or a bivalent, saturated or unsaturated, straight or branched C₁₋₁₂hydrocarbon chain, wherein 0-6 methylene units of L^(x) areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —SO—, —SO₂—, —NHSO₂—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —OC(O)NH—, or—NHC(O)O—, wherein -Cy- is an optionally substituted 5-8 memberedbivalent, saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, oran optionally substituted 8-10 membered bivalent saturated, partiallyunsaturated, or aryl bicyclic ring having 0-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, the L^(x) group of formula IIb is a valencebond. In other embodiments, L^(x) a bivalent, saturated C₁₋₁₂hydrocarbon chain, wherein 0-6 methylene units of L^(x) areindependently replaced by -Cy-, —O—, —NH—, —S—, —OC(O)—, —C(O)O—,—C(O)—, —C(O)NH—, or —NHC(O)—, wherein -Cy- is an optionally substituted3-8 membered bivalent, saturated, partially unsaturated, or aryl ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or an optionally substituted 8-10 membered bivalent saturated,partially unsaturated, or aryl bicyclic ring having 0-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In still otherembodiments, L^(x) a bivalent, saturated C₁₋₆ alkylene chain, wherein0-3 methylene units of L^(x) are independently replaced by -Cy-, —O—,—NH—, —S—, —OC(O)—, —C(O)O—, —C(O)—, —C(O)NH—, or —NHC(O)—.

In certain embodiments, L^(x) is -Cy- (i.e. a C₁ alkylene chain whereinthe methylene unit is replaced by -Cy-), wherein -Cy- is an optionallysubstituted 3-8 membered bivalent, saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. According to one aspect of the present invention,-Cy- is an optionally substituted bivalent aryl group. According toanother aspect of the present invention, -Cy- is an optionallysubstituted bivalent phenyl group. In other embodiments, -Cy- is anoptionally substituted 5-8 membered bivalent, saturated carbocyclicring. In still other embodiments, -Cy- is an optionally substituted 5-8membered bivalent, saturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy-groups include bivalent rings selected from phenyl, pyridyl,pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

In certain embodiments, the group of formula IIb is —O—, —S—, —NH—, or—C(O)O—. In other embodiments, the L^(x) group of formula IIb is -Cy-,—C(O)—, —C(O)NH—, —NHC(O)—, —NH—O—, or —O-Cy-CH₂NH—O—. In still otherembodiments, the L^(x) group of formula IIb is any of —OCH₂—,—OCH₂C(O)—, —OCH₂CH₂C(O)—, —OCH₂CH₂O—, —OCH₂CH₂S—, —OCH₂CH₂C(O) O—,—OCH₂CH₂NH—, —OCH₂CH₂NHC(O)—, —OCH₂CH₂C(O)NH—, and —NHC(O)CH₂CH₂C(O)O—.According to another aspect, the group of formula IIb is any of—OCH₂CH₂NHC(O)CH₂CH₂C(O)O—, —OCH₂CH₂NHC(O)CH₂OCH₂C(O) O—,—OCH₂CH₂NHC(O)CH₂OCH₂C(O)NH—, —CH₂C(O)NH—, —CH₂C(O)NHNH—, or—OCH₂CH₂NHNH—. Exemplary L^(x) groups of formula IIb include any ofthose depicted in any of Tables 1 through 25.

According to another aspect of the present invention, a functional groupformed by the -L^(x)-R^(x) moiety of formula IIb is optionallyprotected. Thus, in certain embodiments, the -L^(x)-R^(x) moiety offormula IIb optionally comprises a mono-protected amine, a di-protectedamine, a protected aldehyde, a protected hydroxyl, a protectedcarboxylic acid, or a protected thiol group. Such groups include thosedescribed above with respect to the -L¹-R¹ moiety of formula I.

Exemplary compounds of formula IIa and IIb are set forth in Tables 1through 25.

According to another embodiment, the present invention provides acompound of either of formulae IIIa or IIIb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

Yet another embodiment relates to a compound of either of formulae IVaor IVb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

Yet another embodiment relates to a compound of either of formulae Va orVb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

In certain embodiments, the present invention provides a compound ofeither of formulae VIa or VIb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

In other embodiments, the present invention provides a compound ofeither of formulae VIIa or VIIb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

In still other embodiments, the present invention provides a compound ofeither of formulae VIIIa or VIIIb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

According to another embodiment, the present invention provides acompound of either of formulae IXa or IXb:

or a salt thereof, wherein n, L², R¹, and R² are as defined above anddescribed in classes and subclasses herein, singly and in combination.

According to yet another embodiment, the present invention provides acompound of either of formulae Xa or Xb:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

In certain embodiments, the present invention provides a compound of anyof formulae XIa, XIb, XIc, XId, XIe, or XIf:

or a salt thereof, wherein n, L¹, L², R¹, and R² are as defined aboveand described in classes and subclasses herein, singly and incombination.

In certain embodiments, the present invention provides a compound asdescribed herein, wherein R¹ is a C₁₋₆ aliphatic substituted with —CO₂H.Exemplary compounds include those set forth in Table 1, wherein n is asdescribed in classes and subclasses herein. In certain embodiments, n isselected from 50±10. In other embodiments, n is selected from 80±10,115±10, 180±10, or 225±10.

TABLE 1 Exemplary Compounds

# R^(a) R^(b)  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

  

26

In certain embodiments, the present invention provides a compound asdescribed herein, wherein R¹ is a C₁₋₆ aliphatic substituted withoxazolinyl. Exemplary compounds include those set forth in Table 2,wherein n is as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 2 Exemplary Compounds

# R^(a) R^(b) 27

28

29

In certain embodiments, the present invention provides a compound asdescribed herein, wherein L¹ is a C₁₋₆ alkylene wherein two methyleneunits of L¹ are substituted with —C(O)NH— and —C(O)O—, and R¹ ishydrogen. Exemplary compounds include those set forth in Table 3,wherein n is as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 3 Exemplary Compounds

# R^(a) R^(b) 30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

In certain embodiments, the present invention provides a compound asdescribed herein, wherein L¹ is a C₁₋₆ alkylene wherein three methyleneunits of L¹ are substituted with —C(O)NH—, —O—, and —C(O)O—, and R¹ ishydrogen. Exemplary compounds include those set forth in Table 4,wherein n is as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 4 Exemplary Compounds

# R^(a) R^(b)  84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108

109

110

In certain embodiments, the present invention provides a compound asdescribed herein, wherein L¹ is —O— and R¹ is hydrogen. Exemplarycompounds include those set forth in Table 5, wherein n is as describedin classes and subclasses herein. In certain embodiments, n is selectedfrom 50±10. In other embodiments, n is selected from 80±10, 115±10,180±10, or 225±10.

TABLE 5 Exemplary Compounds

# R^(a) R^(b) 111

112

113

114

115

116

117

118

119

120

121

122

    

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

In certain embodiments, the present invention provides a compound asdescribed herein, wherein L¹ is —C(O)— and R¹ is hydrogen. Exemplarycompounds include those set forth in Table 6, wherein n is as describedin classes and subclasses herein. In certain embodiments, n is selectedfrom 50±10. In other embodiments, n is selected from 80±10, 115±10,180±10, or 225±10.

TABLE 6 Exemplary Compounds

# R^(a) R^(b) 138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

In certain embodiments, the present invention provides a compound asdescribed herein, wherein -L¹-R¹ moiety of formula I comprises aprotected aldehyde. Exemplary compounds include those set forth in Table7, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 7 Exemplary Compounds

# R^(a) R^(b) 166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises anacetylene moiety. Exemplary compounds include those set forth in Table8, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 8 Exemplary Compounds

# R^(a) R^(b) 186

187

188

189

190

191

192

193

194

195

196

197

198

199

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises aprotected acetylene moiety. Exemplary compounds include those set forthin Table 9, wherein n is as described in classes and subclasses herein.In certain embodiments, n is selected from 50±10. In other embodiments,n is selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 9 Exemplary Compounds

# R^(a) R^(b) 200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises —NH₂.Exemplary compounds include those set forth in Table 10, wherein n is asdescribed in classes and subclasses herein. In certain embodiments, n isselected from 50±10. In other embodiments, n is selected from 80±10,115±10, 180±10, or 225±10.

TABLE 10 Exemplary Compounds

# R^(a) R^(b) 228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

248

249

250

251

252

253

254

255

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises aprotected amino group. Exemplary compounds include those set forth inTable 11, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 11 Exemplary Compounds

# R^(a) R^(b) 256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises an azide.Exemplary compounds include those set forth in Table 12, wherein n is asdescribed in classes and subclasses herein. In certain embodiments, n isselected from 50±10. In other embodiments, n is selected from 80±10,115±10, 180±10, or 225±10.

TABLE 12 Exemplary Compounds

# R^(a) R^(b) 291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises anepoxide. Exemplary compounds include those set forth in Table 13,wherein n is as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 13 Exemplary Compounds

# R^(a) R^(b) 322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the R¹ moiety of formula I comprises adetectable moiety. Exemplary compounds include those set forth in Table14, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 14 Exemplary Compounds

# R^(a) R^(b) 345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₈ memberedalkylene wherein three methylene units of L¹ are replaced by —C(O)NH—,—NHC(O)—, and —NH—, and R¹ is hydrogen. Exemplary compounds includethose set forth in Table 15, wherein n is as described in classes andsubclasses herein. In certain embodiments, n is selected from 50±10. Inother embodiments, n is selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 15 Exemplary Compounds

# R^(a) R^(b) 379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₁₀ memberedalkylene wherein four methylene units of L¹ are replaced by —C(O)NH—,—NHC(O)—, —O—, and —NH—, and R¹ is hydrogen. Exemplary compounds includethose set forth in Table 16, wherein n is as described in classes andsubclasses herein. In certain embodiments, n is selected from 50±10. Inother embodiments, n is selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 16 Exemplary Compounds

# R^(a) R^(b) 407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₆ memberedalkylene wherein two methylene units of L¹ are replaced by —C(O)NH— and—NH—, and R¹ is hydrogen. Exemplary compounds include those set forth inTable 17, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 17 Exemplary Compounds

# R^(a) R^(b) 435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₆ memberedalkylene wherein two methylene units of L¹ are replaced by —NH— and R¹is hydrogen. Exemplary compounds include those set forth in Table 18,wherein n is as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 18 Exemplary Compounds

# R^(a) R^(b) 463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₆ memberedalkylene wherein two methylene units of L¹ are replaced by —O— and —NH—,and R¹ is hydrogen. Exemplary compounds include those set forth in Table19, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 19 Exemplary Compounds

# R^(a) R^(b) 492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₆ memberedalkylene wherein one methylene unit of L¹ is replaced by —O— and R¹ is—CN. Exemplary compounds include those set forth in Table 20, wherein nis as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 20 Exemplary Compounds

# R^(a) R^(b) 520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

In certain embodiments, the present invention provides a compound asdescribed herein, wherein the L¹ moiety of formula I is a C₁₋₆ memberedalkylene wherein one methylene unit of L¹ is replaced by —S— and R¹ is—H. Exemplary compounds include those set forth in Table 21, wherein nis as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 21 Exemplary Compounds

# R^(a) R^(b) 545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

In certain embodiments, the present invention provides a compound asdescribed herein, wherein R¹ is a protected thiol moiety. Exemplarycompounds include those set forth in Table 22, wherein n is as describedin classes and subclasses herein. In certain embodiments, n is selectedfrom 50±10. In other embodiments, n is selected from 80±10, 115±10,180±10, or 225±10.

TABLE 22 Exemplary Compounds

# R^(a) R^(b) 573

574

575

576

In certain embodiments, the present invention provides a compound asdescribed herein, wherein R¹ is C₁₋₆ aliphatic and, in certainembodiments, alkenyl. Exemplary compounds include those set forth inTable 23, wherein n is as described in classes and subclasses herein. Incertain embodiments, n is selected from 50±10. In other embodiments, nis selected from 80±10, 115±10, 180±10, or 225±10.

TABLE 23 Exemplary Compounds

# R^(a) R^(b) 577

578

579

580

In certain embodiments, the present invention provides a compound asdescribed herein, wherein R¹ is a bicyclic, partially unsaturated 7membered ring. Exemplary compounds include those set forth in Table 24,wherein n is as described in classes and subclasses herein. In certainembodiments, n is selected from 50±10. In other embodiments, n isselected from 80±10, 115±10, 180±10, or 225±10.

TABLE 24 Exemplary Compounds

# R^(a) R^(b) 581

582

583

584

In certain embodiments, the present invention provides a compoundselected from those set forth in Table 25, wherein n is as described inclasses and subclasses herein. In certain embodiments, n is selectedfrom 50±10. In other embodiments, n is selected from 80±10, 115±10,180±10, or 225±10.

TABLE 25 Exemplary Compounds

# R^(a) R^(b) 585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

4. General Methods of Providing the Present Compounds

Compounds of this invention may be prepared in general by syntheticmethods known to those skilled in the art for analogous compounds and asillustrated by the general schemes and the preparative examples thatfollow. In certain embodiments, compounds of the present invention areprepared by methods as described in detail in United States patentapplication entitled “Heterobifunctional poly(ethylene glycol) and UsesThereof” filed Oct. 24, 2005, and given Ser. No. 11/256,735, theentirety of which is hereby incorporated herein by reference.

Scheme I above shows a general method for preparing compounds of thepresent invention. At step (a), the polymerization initiator is treatedwith a suitable base to form 2. A variety of bases are suitable for thereaction at step (a). Such bases include, but are not limited to,potassium naphthalenide, diphenylmethyl potassium, triphenylmethylpotassium, and potassium hydride. At step (b), the resulting anion istreated with ethylene oxide to form the polymer 3. Polymer 3 can betransformed at step (d) to a compound of formula I directly byterminating the living polymer chain-end of 3 with a suitablepolymerization terminator to afford a compound of formula I.Alternatively, polymer 3 may be quenched at step (c) to form thehydroxyl compound 4. Compound 4 is then derivatized to afford a compoundof formula I by methods known in the art.

One of ordinary skill in the art will recognize that the derivatizationof a compound of formula 4 to form a compound of formula I may beachieved in a single step or via a multi-step process. For example, thehydroxyl group of formula 4 can be converted to a suitable leaving groupwhich is then displaced by a nucleophile to form a compound of formulaI. Suitable leaving groups are well known in the art, e.g., see,“Advanced Organic Chemistry,” Jerry March, 5^(th) Ed., pp. 351-357, JohnWiley and Sons, N.Y. Such leaving groups include, but are not limitedto, halogen, alkoxy, sulphonyloxy, optionally substitutedalkylsulphonyloxy, optionally substituted alkenylsulfonyloxy, optionallysubstituted arylsulfonyloxy, and diazonium moieties. Examples ofsuitable leaving groups include chloro, iodo, bromo, fluoro,methanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy,nitro-phenylsulfonyloxy(nosyloxy), and bromo-phenylsulfonyloxy(brosyloxy).

According to an alternate embodiment, the suitable leaving group may begenerated in situ within a reaction medium. For example, a leaving groupmay be generated in situ from a precursor of that compound wherein saidprecursor contains a group readily replaced by said leaving group insitu.

Derivatization of the hydroxyl group of formula 4 can be achieved usingmethods known to one of ordinary skill in the art to obtain a variety ofcompounds. For example, said hydroxyl group may be transformed to aprotected hydroxyl group, or, alternatively, to a suitable leavinggroup. Hydroxyl protecting groups are well known and include thosedescribed above and herein. Such transformations are known to oneskilled in the art and include, among others, those described herein.

An exemplary transformation includes coupling of the hydroxyl group offormula 4 with an acid to form an ester thereof. Once of ordinary skillin the art would recognize that this transformation would result incompounds of formula I wherein L² is a bivalent, saturated orunsaturated, straight or branched C₁₋₁₂ alkylene chain, as defined anddescribed herein, wherein the terminal methylene group is replaced by—C(O)O—. Such coupling reactions are well known in the art. In certainembodiments, the coupling is achieved with a suitable coupling reagent.Such reagents are well known in the art and include, for example, DCCand EDC, among others. In other embodiments, the carboxylic acid moietyis activated for use in the coupling reaction. Such activation includesformation of an acyl halide, use of a Mukaiyama reagent, and the like.These methods, and others, are known to one of ordinary skill in theart, e.g., see, “Advanced Organic Chemistry,” Jerry March, 5^(th) Ed.,pp. 351-357, John Wiley and Sons, N.Y.

In certain embodiments, the R²-L²- group of formula I is incorporated ateither of steps (b) or (e) by derivatization of the hydroxyl group offormula 4 via Mitsunobu coupling. The Mitsunobu reaction is a mildmethod for achieving formal substitution of the hydroxyl group usingazodicarboxylic esters/amides and triphenylphosphine (TPP) ortrialkylphosphines or phosphites. In addition, other azo compounds havebeen developed as alternatives to the traditional azodicarboxylic estersdiethylazodicarboxylate (DEAD) and diisopropylazodicarboxylate (DIAD).These include dibenzyl azodicarboxylate (DBAD),N,N,N′,N′-tetramethylazodicarbonamide (TMAD), and dipiperidylazodicarboxylate (DPAD). Mitsunobu coupling provides access to terminalgroups including, but not limited to, halides, azide, amines, esters,ethers, thioethers and isothiocyanates. Accordingly, it will beappreciated that a variety of compounds of formula I are obtained by thederivatization of the hydroxyl group of formula 4 by Mitsunobu reaction.

In certain embodiments, the polymerization terminating agent is one thatis capable of Mitsunobu coupling. These include optionally substitutedphenols, optionally substituted thiophenols, cyclic imides, carboxylicacids, azide, and other reagents capable of Mitsunobu coupling. SuchMitsunobu terminating agents include, but are not limited to, those setforth in Table 26, below.

TABLE 26 Representative Mitsunobu Polymerization Terminating Agents

M-1

M-2

M-3

M-4

M-5

M-6

M-7

M-8

M-9

M-10

M-11

M-12

M-13

M-14

M-15

M-16

M-17

M-18

M-19

M-20

M-21

M-22

M-23

M-24

M-25

M-26

M-27

M-28

M-29

M-30

M-31

M-32

M-33

M-34

M-35

M-36

M-37

M-38

M-39

M-40

M-41

M-42

M-43

M-44

M-45

M-46

M-47

M-48

M-49

M-50

M-51

M-52

M-53

M-54

M-55

M-56

M-57

M-58

M-59 NaBr M-60 NaI M-61 H—N₃ M-62 Na—N₃ M-63

M-64

M-65

M-66

M-67

M-68

M-69

M-70

M-71

M-72

M-73

M-74

M-75

M-76

M-77

M-78

M-79

M-80

M-81

M-82

M-83

M-84

M-85

M-86

In other embodiments, the R²-L²- group of formula I is incorporated byderivatization of the hydroxyl group of formula 4 via anhydridecoupling. One of ordinary skill in the art would recognize thatanhydride polymerization terminating agents containing an azide, analdehyde, a protected hydroxyl, an alkyne, and other groups, may be usedto incorporate said azide, said aldehyde, said protected hydroxyl, saidalkyne, and other groups into the R²-L²- group of compounds of formulaI. It will also be appreciated that such anhydride polymerizationterminating agents are also suitable for terminating the living polymerchain-end of a compound of formula 3. Such anhydride polymerizationterminating agents include, but are not limited to, those set forth inTable 27, below.

TABLE 27 Representative Anhydride Polymerization Terminating Agents

A-1

A-2

A-3

A-4

A-5

A-6

A-7

A-8

A-9

A-10

A-11

A-12

A-13

In other embodiments, the R²-L²-group of formula I is incorporated byderivatization of the hydroxyl group of formula 4 via reaction with apolymerization terminating agent having a suitable leaving group. Itwill also be appreciated that such polymerization terminating agents arealso suitable for terminating the living polymer chain-end of a compoundof formula 3. Examples of these polymerization terminating agentsinclude, but are not limited to, those set forth in Table 28, below.

TABLE 28 Representative Polymerization Terminating Agents

L-1

L-2

L-3

L-4

L-5

L-6

L-7

L-8

L-9

L-10

L-11

L-12

L-13

L-14

L-15

L-16

L-17

L-18

L-19

L-20

L-21

L-22

L-23

L-24

L-25

L-26

L-27

L-28

L-29

L-30

L-31

L-32

L-33

L-34

L-35

L-36

L-37

L-38

L-39

L-40

L-41

L-42

L-43

L-44

wherein each L is a suitable leaving group as defined above and inclasses and subclasses as described above and herein.

One of ordinary skill in the art will recognize that certain of theterminating groups depicted in Tables 26, 27, and 28 comprise protectedfunctional groups. It will be appreciated that these protecting groupsare optionally removed to form compounds of the present invention.Methods for the deprotection of functional groups are well known to oneof ordinary skill in the art and include those described in detail inGreene (1999).

Although certain exemplary embodiments are depicted and described aboveand herein, it will be appreciated that compounds of the invention canbe prepared according to the methods described generally above usingappropriate starting materials by methods generally available to one ofordinary skill in the art. Additional embodiments are exemplified inmore detail herein.

5. Uses, Methods, and Compositions

As discussed above, the present invention provides bifunctional PEG's,intermediates thereto, and methods of preparing the same. Suchfunctionalized PEG's are useful for a variety of purposes in thepharmaceutical and biomedical fields. Such uses include using thebifunctional PEG's of the present invention in the process of PEGylatingother molecules or substrates. Accordingly, another embodiment of thepresent invention provides a molecule or substrate conjugation with acompound of the present invention. The term “PEGylation,” as usedherein, is used interchangeably with the term “conjugation”. Thus, theproduct of PEGylation is known as a “conjugate.”

For example, U.S. Pat. No. 6,797,257 describes imaging agents preparedby PEGylating gadolinium oxide albumin microspheres. U.S. Pat. Nos.6,790,823 and 6,764,853 describe the PEGylation of proteins bycovalently bonding through amino acid residues via a reactive group,such as, a free amino or carboxyl group. Reactive groups are those towhich an activated polyethylene glycol molecule may be bound. Amino acidresidues having a free amino group include lysine residues. N-terminalamino acid residues; i.e. those having a free carboxyl group, includeaspartic acid residues, glutamic acid residues, and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecule(s).

Another aspect of the invention provides a method of PEGylating aprimary or secondary label, a dye, or another detectable moiety forbiosensors, bioassays, biorecognition, detection, proteomics, genomics,microarray, and other molecular biological applications. Thus, incertain embodiments, the present invention provides a detectable moietyconjugated with a compound of the present invention. Such PEGylation maybe carried out by covalent linking of one PEG functionality to thedetectable moiety or through coordination of a PEG functionality (e.g.thiol, amine, alcohol, carboxylic acid) to the detectable moiety. Theopposite PEG end group can be further linked to targeting groups,permeation enhancers, proteins, sugars, DNA, RNA, cells, viruses, orother biomolecules for targeted delivery or recognition. Such labels ordetectable moieties include but are not limited to organic and inorganicdyes, semiconducting nanoparticles (e.g. CdSe, CdS, CdSe/ZnS, ZnSe, PbSenanoparticles), magnetic nanoparticles (e.g. Co, FePt, Fe₃O₄, Fe₂O₃nanoparticles), or other metal nanoparticles (e.g. Au nanoparticles).For representative examples of nanoparticle PEGylation see Takae, S.;Akiyama, Y.; Otsuka, H.; Nakamura, T.; Nagasaki, Y.; Kataoka, K. “Liganddensity effect on biorecognition by PEGylated gold nanoparticles:regulated interaction of RCA120 lectin with lactose installed to thedistal end of tethered PEG strands on gold surface” Biomacromolecules2005, 6, 818-824; Ishii, T.; Sunaga, Y.; Otsuka, H.; Nagasaki, Y.;Kataoka, K. “Preparation of water soluble CdS quantum dots stabilized byfunctional poly(ethylene glycol) and its application for bioassay” J.Photopolym. Sci. Technol. 2004, 17, 95-98; Otsuka, H.; Akiyama, Y.;Nagasaki, Y.; Kataoka, K. “Quantitative and Reversible Lectin-InducedAssociation of Gold Nanoparticles Modified withα-Lactosyl-ω-mercapto-poly(ethylene glycol)” J. Am. Chem. Soc. 2001,123, 8226-8230; Akerman, M. E.; Chan, W. C. W.; Laakkonen, P.; Bhatia,S. N.; Ruoslahti, E. R. “Nanocrystal targeting in vivo” P. Natl. Acad.Sci. USA 2002, 99, 12617-12621; Skaff, H.; Emrick, T. “A Rapid Route toAmphiphilic Cadmium Selenide Nanoparticles Functionalized withPoly(ethylene glycol)” Chem. Comm., 2003, 1, 52-53.

Accordingly, another aspect of the present invention provides a methodof PEGylating a biomolecule with a compound of formula I as describedgenerally above and in classes and subclasses defined above and herein.Thus, in certain embodiments, the present invention provides abiomolecule conjugated with a compound of the present invention. Incertain embodiments, the present invention provides a method ofPEGylating a therapeutic or a therapeutic carrier such as a protein, acell, a virus particle, a plasmid, an oligopeptide, an oligonucleotide(e.g. siRNA, miRNA, aptamer), small molecule drug, a liposome, apolymersome, a polymer microshere, or a lipid emulsion with a compoundof formula I as described generally above and in classes and subclassesdefined above and herein. According to another aspect, the presentinvention provides a method for PEGylating a substrate. Such PEGylationmay be carried out by covalent linking of a terminal PEG functionalityto the substrate or using any number of bioconjugation techniques.

The bifunctional PEG's of the present invention are also useful forlinking two biomolecules together wherein said biomolecules are the sameor different from each other. For example, one terminus of the presentcompounds may be linked to a surface, another polymer, therapeutic,therapeutic carrier, protein, cell, virus particle, a plasmid,oligopeptide, oligonucleotide (e.g. siRNA, miRNA, aptamer), smallmolecule drug, liposome, polymersome, polymer microshere, lipidemulsion, or a detectable moiety and the other terminus of the presentcompounds may be linked to a surface, targeting group, permeationenhancer, growth factor, protein, sugar, DNA, RNA, cell, virus,diagnostic agent, or a detectable moiety. Accordingly, the presentinvention also provides a method for linking two biomolecules togetherwherein said method comprises coupling one terminus of a compound offormula I to a first biomolecule then coupling the other terminus of acompound of formula I to a second molecule, wherein the first and secondbiomolecules may be the same or different from each other.

Accordingly, one aspect of the present invention provides a method ofPEGylating a protein therapeutic with a compound of formula I asdescribed generally above and in classes and subclasses defined aboveand herein. Thus, in certain embodiments, the present invention providesa protein therapeutic conjugated with a compound of the presentinvention. Such PEGylation may be carried out by covalent linking of onePEG functionality to the protein using any number of bioconjugationtechniques. The opposite PEG end group can be further linked totargeting groups, permeation enhancers, proteins, sugars, DNA, RNA,cells, viruses, dyes, detectable moieties, labels or other biomoleculesfor targeted delivery, biorecognition, or detection. For representativeexamples of PEGylating a protein see Harris, J. M.; Chess, R. B. “Effectof PEGylation on Pharmaceuticals” Nat. Rev. Drug. Discov. 2003, 2,214-221; Kozlowski, A.; Harris, J. M. “Improvements in proteinPEGylation: pegylated interferons for treatment of hepatitis C” J.Control. Release 2001, 72, 217-224; Koslowski, A.; Charles, S. A.;Harris, J. M. “Development of pegylated interferons for the treatment ofchronic hepatitis C” Biodrugs 2001, 15, 419-429; Harris, J. M.; Martin,N. E.; Modi, M. “Pegylation: a novel process for modifyingpharmacokinetics” Clin. Pharmacokinet. 2001, 40, 539-551; Roberts, M.J.; Bentley, M. D.; Harris, J. M. “Chemistry for peptide and proteinPEGylation” Adv. Drug Deliver. Rev. 2002, 54, 459-476.

Another aspect of the present invention provides a method of PEGylatinga small molecule drug with a compound of formula I as describedgenerally above and in classes and subclasses defined above and herein.Thus, in certain embodiments, the present invention provides a smallmolecule drug conjugated with a compound of the present invention, alsoreferred to as a “drug-polymer conjugate.” Such PEGylation may becarried out by covalent linking of one PEG functionality to the smallmolecule drug using any number of bioconjugation techniques. Theopposite PEG end group can be further linked to targeting groups,permeation enhancers, proteins, sugars, DNA, RNA, cells, viruses, dyes,detectable moieties, labels or other biomolecules for targeted delivery,biorecognition, or detection. For representative examples of

PEGylating a small molecule drug see Greenwald, R. B. “PEG drugs: anoverview” J. Control. Release 2001, 74, 159-171; Caliceti, P.;Monfardini, C.; Sartore, L.; Schiavon, O.; Baccichetti, F.; Carlassare,F.; Veronese, F. M. “Preparation and properties of monomethoxypoly(ethylene glycol) doxorubicin conjugates linked by an amino acid ora peptide as spacer” Il Farmaco 1993, 48, 919-932; Fleming, A. B.;Haverstick, K.; Saltzman, W. M. “In vitro cytotoxicity and in vivodistribution after direct delivery of PEG-camptothecin conjugates to therat brain” Bioconjug. Chem. 2004, 15, 1364-1375.

Yet another aspect of the present invention provides a drug-polymerconjugate comprising a compound of formula I and a pharmaceuticallyactive agent. In still another aspect of the present invention,pharmaceutically acceptable compositions are provided, wherein thesecompositions comprise a drug-polymer conjugate as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain embodiments, such compositions optionally furthercomprise one or more additional therapeutic agents.

One of ordinary skill in the art would recognize that the presentcompounds are useful for the PEGylation of small molecule drugs. Smallmolecule drugs suitable for PEGylation with the present compoundsinclude, but are not limited to, those having a functional groupsuitable for covalently linking to the bifunctional PEG's of the presentinvention. Such drugs include, without limitation, chemotherapeuticagents or other anti-proliferative agents including taxanes (Taxol andtaxotere derivatives), camptothecin, alkylating drugs (mechlorethamine,chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites(Methotrexate), purine antagonists and pyrimidine antagonists(6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), spindlepoisons (Vinblastine, Vincristine, Vinorelbine, Paclitaxel),podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics(Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine,Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes(Asparaginase), angiogenesis inhibitors (Avastin) and hormones(Tamoxifen, Leuprolide, Flutamide, and Megestrol), Gleevec,dexamethasone, and cyclophosphamide. For a more comprehensive discussionof updated cancer therapies see, http://www.nci.nih.gov/, a list of theFDA approved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Other examples of small molecule drugs that may be PEGylated with thecompounds of this invention include treatments for Alzheimer's Diseasesuch as Aricept® and Excelon®; treatments for Parkinson's Disease suchas L-DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine,pergolide, trihexephendyl, and amantadine; agents for treating MultipleSclerosis (MS) such as beta interferon (e.g., Avonex® and Rebie),Copaxone®, and mitoxantrone; treatments for asthma such as albuterol andSingulair®; agents for treating schizophrenia such as zyprexa,risperdal, seroquel, and haloperidol; anti-inflammatory agents such ascorticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide,and sulfasalazine; immunomodulatory and immunosuppressive agents such ascyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons,corticosteroids, cyclophosphamide, azathioprine, and sulfasalazine;neurotrophic factors such as acetylcholinesterase inhibitors, MAOinhibitors, interferons, anti-convulsants, ion channel blockers,riluzole, and anti-Parkinsonian agents; agents for treatingcardiovascular disease such as beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins; agents for treatingliver disease such as corticosteroids, cholestyramine, interferons, andanti-viral agents; agents for treating blood disorders such ascorticosteroids, anti-leukemic agents, and growth factors; and agentsfor treating immunodeficiency disorders such as gamma globulin.

Another aspect of the present invention provides a method of PEGylatinga virus with a compound of formula I as described generally above and inclasses and subclasses defined above and herein. Thus, in certainembodiments, the present invention provides a virus conjugated with acompound of the present invention. Such PEGylation may be carried out bycovalent linking of one PEG functionality to the virus using any numberof bioconjugation techniques. The opposite PEG end group can be furtherlinked to targeting groups, permeation enhancers, proteins, sugars, DNA,RNA, cells, viruses, dyes, detectable moieties, labels or otherbiomolecules for targeted delivery, biorecognition, or detection. Forrepresentative examples of virus PEGylation see Gupta, S. S.; Kuzelka,J.; Singh, P.; Lewis, W. G.; Manchester, M.; Finn, M. G. “AcceleratedBioorthogonal Conjugation: A Practical Method for the Ligation ofDiverse Functional Molecules to a Polyvalent Virus Scaffold” Bioconjug.Chem. 2005, 16, 1572-1579; Raja, K. S.; Wang, Q.; Gonzalez, M. J.;Manchester, M.; Johnson, J. E.; Finn, M. G. “Hybrid Virus-PolymerMaterials. 1. Synthesis and Properties of PEG-Decorated Cowpea MosaicVirus” Biomacromolecules 2003, 4, 472-476; Oh, I. K.; Mok, H.; Park, T.G. “Folate Immobilized and PEGylated Adenovirus for Retargeting to TumorCells” Bioconjugate Chem. ASAP Article (Published online Apr. 14, 2006)

Yet another aspect of the present invention provides a method ofPEGylating therapeutic carriers such as liposomes, polymersomes,microspheres, capsules, or lipid emulsions with a compound of formula Ias described generally above and in classes and subclasses defined aboveand herein. Thus, in certain embodiments, the present invention providesa therapeutic carrier conjugated with a compound of the presentinvention. Such PEGylation may be carried out by covalent linking of onePEG functionality to the therapeutic carrier using any number ofbioconjugation techniques or by the non-covalent incorporation of aPEGylated molecule (e.g. lipid, phospholipid, or polymer) into thecarrier. The opposite PEG end group can be further linked to targetinggroups, permeation enhancers, proteins, sugars, DNA, RNA, cells,viruses, dyes, detectable moieties, labels or other biomolecules fortargeted delivery, biorecognition, or detection. For representativeexamples of PEGylating therapeutic carriers see Lukyanov, A. N.;Elbayoumi, T. A.; Chakilam, A. R.; Torchilin, V. P. “Tumor-targetedliposomes: doxorubicin-loaded long-circulating liposomes modified withanti-cancer antibody” J. Control. Release 2004, 100, 135-144; Forssen,E.; Willis, M. “Ligand-targeted liposomes” Adv. Drug Del. Rev. 1998, 29,249-271; Koning, G. A.; Schiffelers, R. M.; Wauben, M. H. M.; Kok, R.J.; Mastrobattista, E.; Molema, G.; ten Hagen, T. L. M.; Storm, G.“Targeting of Angiogenic Endothelial Cells at Sites of Inflammation byDexamethasone Phosphate-Containing RGD Peptide Liposomes InhibitsExperimental Arthritis” Arthritis Rheum. 2006, 54, 1198-1208; Torchilin,V. P. “Structure and design of polymeric surfactant-based drug deliverysystems” J. Control. Release 2001, 73, 137-172.

Another aspect of the present invention provides a method of PEGylatinga cell with a compound of formula I as described generally above and inclasses and subclasses defined above and herein. Thus, in certainembodiments, the present invention provides a cell conjugated with acompound of the present invention. Such PEGylation may be carried out bycovalent linking of one PEG functionality to the cell using any numberof bioconjugation techniques. The opposite PEG end group can be furtherlinked to targeting groups, permeation enhancers, proteins, sugars, DNA,RNA, cells, viruses, dyes, detectable moieties, labels or otherbiomolecules for targeted delivery, biorecognition, or detection. SeeScott, M. D.; Chen, A. M. “Beyond the red cell: pegylation of otherblood cells and tissues” Transfus. Clin. Biol. 2004, 11, 40-46.

Another aspect of the present invention provides a method of PEGylatingthe surface of a natural or synthetic material or biomaterial with acompound of formula I as described generally above and in classes andsubclasses defined above and herein. Thus, in certain embodiments, thepresent invention provides a surface conjugated with a compound of thepresent invention. Such PEGylation may be carried out by covalentlinking of one PEG functionality to the surface using any number ofbioconjugation techniques or through non-covalent interactions with PEGor the PEG end-groups. Such surface PEGylation generally enhancesanti-fouling properties of the material and can reduce the foreign-bodyresponse of injectable or implantable biomaterials. For representativeexamples of Bergstrom, K.; Holmberg, K.; Safranj, A.; Hoffman, A. S.;Edgell, M. J.; Kozlowski, A.; Hovanes, B. A.; Harris, J. M. “Reductionof fibrinogen adsorption on PEG-coated polystyrene surfaces” J. Biomed.Mater. Res. 1992, 26, 779-790; Vladkova, T.; Krasteva, N.; Kostadinova,A.; Altankov, G. “Preparation of PEG-coated surfaces and a study fortheir interaction with living cells” J. Biomater. Sci. Polym. Ed. 1999,10, 609-620.

Another aspect of the present invention provides a method of linkingmolecules or biomolecules to a synthetic or natural surface with acompound of formula I as described generally above and in classes andsubclasses defined above and herein. Such PEGylation may be carried outby covalent linking of one PEG functionality to the surface using anynumber of bioconjugation techniques or through non-covalent interactionswith PEG or the PEG end-groups. The opposite PEG end group can befurther linked to proteins, sugars, DNA, RNA, cells, viruses, dyes,detectable moieties, labels or other biomolecules for biorecognitionand/or detection. For representatives examples of using PEGylatedsurface linkers see Otsuka, H.; Nagasaki, Y.; Kataoka, K.“Characterization of aldehyde-PEG tethered surfaces: influence of PEGchain length on the specific biorecognition” Langmuir 2004, 20,11285-11287; Muñoz, E. M.; Yu, H.; Hallock, J.; Edens, R. E.; Linhardt,R. J. “Poly(ethylene glycol)-based biosensor chip to studyheparin-protein interactions” Anal. Biochem. 2005, 343, 176-178;Metzger, S. W.; Natesan, M.; Yanavich, C.; Schneider, J.; Leea, G. U.“Development and characterization of surface chemistries formicrofabricated biosensors” J. Vac. Sci. Technol. A 1999, 17, 2623-2628;Hahn, M. S.; Taite, L. J.; Moon, J. J.; Rowland, M. C.; Ruffino, K. A.;West, J. L. “Photolithographic patterning of polyethylene glycolhydrogels” Biomaterials 2006, 27, 2519-2524; Veiseh, M.; Zareie, M. H.;Zhang, M. “Highly Selective Protein Patterning on Gold-SiliconSubstrates for Biosensor Applications” Langmuir, 2002, 18, 6671-6678.

Another aspect of the present invention provides a method ofincorporating PEG into a hydrogel with a compound of formula I asdescribed generally above and in classes and subclasses defined aboveand herein. Thus, in certain embodiments, the present invention providesa hydrogel conjugated with a compound of the present invention. SuchPEGylation may be carried out by the reaction of one PEG functionalityfor incorporation into the hydrogel matrix or through non-covalentinteraction of the hydrogel and PEG or the PEG end-groups. The oppositePEG end group can be further linked to proteins, growth factors,antibodies, oligopeptides, sugars, DNA, RNA, cells, viruses, dyes,detectable moieties, labels or other biomolecules to promote celladhesion and growth, for biorecognition, or detection. For examples ofproducing hydrogels from functional PEGs see Kim, P.; Kim, D. H.; Kim,B.; Choi, S. K.; Lee, S. H.; Khademhosseini, A.; Langer, R.; Suh, K. Y.“Fabrication of nanostructures of polyethylene glycol for applicationsto protein adsorption and cell adhesion” Nanotechnology, 2005, 16,2420-2426; Raeber, G. P.; Lutolf, M. P; Hubbell, J. A. “MolecularlyEngineered PEG Hydrogels: A Novel Model System for ProteolyticallyMediated Cell Migration” Biophys. J. 2005, 89, 1374-1388; Quick, D. J.;Anseth, K. S. “DNA delivery from photocrosslinked PEG hydrogels:encapsulation efficiency, release profiles, and DNA quality” J. Control.Release 2004, 96, 341-351.

Another aspect of the present invention provides a method of producingblock and graft copolymers of PEG using a compound of formula I asdescribed generally above and in classes and subclasses defined aboveand herein. Thus, in certain embodiments, the present invention providesa block or graft copolymer comprising a compound of the presentinvention. PEGs of formula I which possess appropriate reactivefunctionality may serve as macroinitiators of cyclic esters (e.g.caprolactone, lactide, glycolide), cyclic ethers, cyclic phosphazenes,N-carboxyanhydrides (NCAs), or vinyl monomers (e.g.N-isopropylacrylamide, methyl acrylate, styrene) to synthesize blockcopolymers for use as micellar therapeutic carriers. One or both PEGfunctionalities can be used to initiate or mediate the growth ofadditional polymer blocks. In cases where a single PEG functionalityserves as an initiator, the opposite PEG end group can be further linkedto targeting groups, permeation enhancers, proteins, sugars, DNA, RNA,cells, viruses, dyes, detectable moieties, labels or other biomoleculesfor targeted delivery, biorecognition, or detection. For representativeexamples of PEG macroinitiators see Akiyama, Y.; Harada, A.; Nagasaki,Y.; Kataoka, K. Macromolecules 2000, 33, 5841-5845; Yamamoto, Y.;Nagasaki, Y.; Kato, Y.; Sugiyama, Y.; Kataoka, K. “Long-circulatingpoly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles withmodulated surface charge” J. Control. Release 2001, 77, 27-38; Bae, Y.;Jang, W. D.; Nishiyama, N.; Fukushima, S.; Kataoka, K. “Multifunctionalpolymeric micelles with folate-mediated cancer cell targeting andpH-triggered drug releasing properties for active intracellular drugdelivery” Mol. Biosyst. 2002, 1, 242-250; Nasongkla, N.; Shuai, X.; Ai,H.; Weinberg, B. D.; Pink, J.; Boothman, D. A.; Gao, J.“cRGD-Functionalized Polymer Micelles for Targeted Doxorubicin Delivery”Angew. Chem. Int. Ed. 2004, 43, 6323-6327.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of thedisorder being treated. The exact amount required will vary from subjectto subject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

EXAMPLES

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, in addition to the Schemes set forth above and othermethods known to one of ordinary skill in the art, can be applied to allcompounds and subclasses and species of each of these compounds, asdescribed herein. Each compound number referenced below corresponds tocompound numbers recited in Tables 1 through 25, supra.

GENERAL METHODS Method A: Polymerization

To a stirred solution of initiator (1 mmol) in anhydrous THF (100 mL)was added a solution of potassium naphthalenide in THF (0.2 M, 5 mL, 1mmol). The resulting solution was then cooled to 0° C. Ethylene oxide(10 g, 227 mmol) was introduced to the alkoxide solution using Schlenktechniques. Upon complete addition of the ethylene oxide, the flask wasbackfilled with Argon, sealed and stirred at room temperature for 24 h.At this point, additional terminating agents were added or the reactionwas quenched with water and methanol followed by the removal of solventunder reduced pressure.

Method B: Purification by Solid Phase Extraction

The viscous liquid containing the desired polymer was loaded onto 100 gsilica gel which was rinsed with 3% MeOH in CHCl₃ (1 L) followed by 10%MeOH in CHCl₃ (1 L) which contained the polymer product. The solvent wasremoved and the resulting liquid was diluted with a minimal amount ofmethanol and precipitated into diethyl ether. A white powder wasisolated following filtration.

Method C: Purification by Liquid Extraction

The viscous liquid containing the desired polymer was dissolved in 100mL water then extracted with CHCl₃ (4×300 mL). The combined organiclayers dried over MgSO₄, and filtered. The solvent was removed and theresulting liquid was diluted with a minimal amount of methanol andprecipitated in to diethyl ether. A white powder was isolated followingfiltration.

Method D: Removal of Benzyl Protecting Groups

To a 250 mL round bottom flask was added 10% palladium hydroxide oncarbon (0.6 g) and methanol (50 mL). Dibenzylamino-polyethylene glycol(10 g) and ammonium formate (2 g) was added and the reaction heated toreflux for 16 hours. The solution was cooled, diluted with chloroform(100 mL) then filtered over celite, then the solvent removed. Theresulting liquid was dissolved in 1 N sodium hydroxide and purifiedaccording to Method C.

Method E: Application of the BOC Protecting Group

To a 250 mL round bottom flask was added amino-polyethyleneglycol-alcohol (10 g) and methanol (150 mL). Di-t-butyldicarbonate (10equiv) and DMAP (1 equiv) was added and the resulting solution stirredat room temperature. The solvent was removed and purified according toMethod B.

Method F: Mitsunobu Coupling

The desired PEG derivative (1 equiv) was dissolved in dichloromethane(˜10 mL/g PEG). Triphenylphosphine (4 equiv) followed by the desiredMitsunobu terminating agent (5 equiv) then DIAD (3 equiv) was added tothe solution then stirred for 8 hours. The solvent was removed andpurified according to Method B.

Method G: Mesylation

The desired PEG derivative (1 equiv) was dissolved in CH₂Cl₂ (˜10 mL/gPEG), and cooled to 0° C. Methanesulfonyl chloride (2 equiv) was addeddropwise via syringe under nitrogen followed by addition oftriethylamine (2.5 equiv). The solution was warmed to room temperatureand stirred for 12 hours. The solvent was removed and the product usedas is or was optionally further purified by Method B.

Method H: Azide Functionalization

The desired PEG derivative (1 equiv) was dissolved in ethanol (˜10 mL/gPEG), and then NaN₃ (10 equiv) was added. The solution was stirred atreflux for 16 hours, allowed to cool, the solvent evaporated andpurified by Method B.

Method I: Removal of the BOC Protecting Group

The desired PEG (1 g) was dissolved in a minimal amount of THF (2 mL)and stirred at room temperature. HCl in dioxane (4M, 5 mL) was added andthe solution stirred under Argon for 6 hours. The solution was pouredinto cold diethyl ether and the polymer product obtained as a whitepowder following filtration.

Method J: Removal of the Oxazoline Protecting Group

The desired PEG (1 g) was dissolved in 10 mL of 3 N HCl (aq) and stirredat reflux for 4 hours. The solution was cooled and purified according toMethod C.

Method K: Application of the Trifluoracetamide Group

The desired amino-PEG derivative (1 equiv) was dissolved in methanol((˜10 mL/g PEG). Ethyl trifluoracetate (3 equiv) was added and theresulting solution stirred at room temperature for 16 h. The solvent wasremoved and purified according to Method B.

Method L: Removal of the THP Protecting Group

The desired PEG derivative (1 equiv) was dissolved in ethanol (˜10 mL/gPEG), and then pyridinium para-toluene sulfonate (PPTS) (3 equiv) wasadded. The solution was stirred at reflux for 16 hours, allowed to cool,the solvent evaporated and purified by Method C.

Method M: Removal of the Furan Protecting Group

The desired PEG derivative was dissolved in toluene (˜10 mL/g PEG) andrefluxed for 4 hours. After allowing the solution to cool, the polymerwas precipitated in to diethyl ether. A white powder was isolatedfollowing filtration.

Example 1

Dibenzylamino-poly(ethylene glycol)-alcohol was prepared according toMethod A and purified according to Method B in 80% yield. ¹H NMR (400MHz, DMSO-d₆, δ) 7.4-7.2 (m, Ar—H), 4.63 (t, CH₂OH), 3.7-3.3 (br-m,—O—CH₂—CH₂—O—). GPC (DMF, PEG standards) M_(n)=10,800; PDI=1.10.

Example 2

Amino-poly(ethylene glycol)-alcohol was prepared according to Method Din 84% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 3.7-3.3 (br-m,—O—CH₂—CH₂—O—), 2.62 (m, —CH₂—NH₂).

Example 3

Boc-amino-poly(ethylene glycol)-alcohol was prepared according to MethodE in 89% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 6.82 (br-s, CH₂—NH—CO—),4.63 (t, CH₂OH), 3.7-3.3 (br-m, —O—CH₂—CH₂—O), 1.40 (s, —C—(CH₃)₃). GPC(DMF, PEG standards) M_(n)=10,100; PDI=1.06.

Example 4

Dibenzylamino-poly(ethylene glycol)-diethylphosphonate was preparedaccording to Method A. After 24 h, vinyl-diethylphosphonate (0.82 g, 5mmol) was added to the reaction using Schlenk techniques. The solutionwas stirred for and additional 12 h at 40° C., allowed to cool, and thesolvent removed. The resulting viscous liquid was purified by solidphase extraction (The liquid was loaded onto 300 mL silica gel which wasrinsed with 3% MeOH in CHCl₃ (1 L) followed by 10% MeOH in CHCl₃ (1 L)which contained the polymer product) then precipitation into colddiethyl ether to give a white powder (7.4 g, 73% yield). ¹H NMR (400MHz, DMSO-d₆, δ) 7.3-7.2 (m, Ar—H), 4.01 (m, CH₃—CH₂—O), 3.7-3.3 (br-m,—O—CH₂—CH₂—) 2.55 (s, Ar—CH₂—N), 1.24 (m, CH₃—CH₂—O). GPC (THF, PEGstandards) M_(n)=7,700; PDI=1.05.

This compound is debenzylated according to Method D to form Compound246.

Example 5

Dibenzylamino-poly(ethylene glycol)-diethylphosphonate (1 g, 0.33 mmol)was dissolved in anhydrous methylene chloride (10 mL). TMS-Br (0.17 mL,1.3 mmol) was added via syringe and the resulting solution stirred atroom temperature for 16 hours. The reaction was quenched with water (1mL, 55 mmol) then the solution precipitated into cold diethyl ether. Theproduct was obtained as a white powder following filtration (0.85 g, 85%yield). ¹H NMR (400 MHz, DMSO-d₆, δ) 7.58, 7.47, 3.94, 3.7-3.3, 2.69.

This compound is debenzylated according to Method D to form Compound248.

Example 6

Tetrahydropyran-poly(ethylene glycol)-propyne was prepared according toMethod A. After 24 h, propargyl bromide (3.9 g, 33 mmol) was added tothe reaction using Schlenk techniques. The solution was stirred for andadditional 12 h at 40° C., allowed to cool, and the solvent removed. Theresidue was purified according to Method B in 74% yield. ¹H NMR (400MHz, DMSO-d₆, δ) 4.55, 4.14, 3.7-3.3, 1.71, 1.61, 1.46. GPC (THF, PEGstandards) M_(n)=2,400; PDI=1.04.

Example 7

Azido-poly(ethylene glycol)-alcohol was prepared according to Method Afollowed by Method B in 80% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 4.57 (t,CH₂OH), 3.7-3.3 (br-m, -0-CH₂—CH₂—). GPC (THF, PEG standards)M_(n)=9,500; PDI=1.05.

Example 8

t-Butyldiphenylsilylpropargyl-poly(ethylene glycol) was preparedaccording to Method A followed by Method B in 59% yield. ¹H NMR (400MHz, DMSO-d₆, δ) 7.62 (m, Ar—H), 7.41 (m, Ar—H), 4.55 (t, CH₂OH),3.7-3.3 (br-m, —O—CH₂—CH₂—O—), 0.91 (s, t-butyl). GPC (THF, PEGstandards) M_(n)=2,700; PDI=1.17.

Example 9

Dibenzylamino-poly(ethylene glycol)-benzoic acid benzyl ester wasprepared according to Method A followed by Method F in 74% yield. ¹H NMR(400 MHz, DMSO-d₆, δ) 7.95, 7.6-7.2, 7.05, 4.15, 3.75, 3.6-3.3, 2.55.GPC (THF, PEG standards) M_(n)=1,800; PDI=1.06.

Example 10

Amino-poly(ethylene glycol)-benzoic acid (Compound 228)was preparedaccording to Method D in 74% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 7.9,7.1, 3.7-3.3.

Example 11

Dibenzylamino-polyethylene glycol-phthalimide was prepared according toMethod A followed by Method F in 73% yield. ¹H NMR (400 MHz, DMSO-d₆, δ)7.85 (m, phthalimide Ar—H), 7.4-7.2 (m, Ar—H), 3.7-3.3 (br-m,—O—CH₂—CH₂—O—). GPC (DMF, PEG standards) M_(n)=10,900; PDI=1.11.

Example 12

Amino-polyethylene glycol-phthalimide was prepared according to Method Din 63% yield. NMR (400 MHz, DMSO-d₆, δ) 7.87, 7.32, 3.7-3.3, 2.66.

Example 13

Hexyne-polyethylene glycol-BOC-aminophenoxy ether was prepared accordingto Method A followed by Method F in 70% yield. ¹H NMR (400 MHz, DMSO-d₆,δ) 7.85 (m, phthalimide Ar—H), 7.35 (d, Ar—H), 6.85 (d, Ar—H), 3.7-3.3(br-m, —O—CH₂—CH₂—O—), 2.14 (m, —CH₂), 1.73 (t, CH₃), 1.61 (q, —CH₂),1.39 (s, —C—(CH₃)₃). GPC (DMF, PEG standards) M_(n)=10,800; PDI=1.10.

Example 14

Hexyne-polyethylene glycol-amine hydrochloride phenoxy ether wasprepared according to Method I in 87% yield. NMR (400 MHz, DMSO-d₆, 6)8.4 (br-s) 7.80 (m, phthalimide Ar—H), 7.37 (d, Ar—H), 6.85 (d, Ar—H),3.7-3.3 (br-m, —O—CH₂—CH₂—O—), 2.13 (m, —CH₂), 1.73 (t, CH₃), 1.61 (q,—CH₂).

Example 15

Tetrahydropyran-poly(ethylene glycol)-phosphonic ester was preparedaccording to Method A. After 24 h, vinyl diethyl phosphonate (3.2 g, 20mmol) was added to the reaction using Schlenk techniques. The solutionwas stirred for and additional 12 h at 40° C., allowed to cool, and thesolvent removed and purified by Method B in 70% yield. ¹H NMR (400 MHz,DMSO-d₆, δ) 4.01, 3.3-3.7, 2.06, 1.71, 1.58, 1.45, 1.22, 1.09. GPC (DMF,PEG standards) M_(n)=6,700; PDI=1.05.

The THP group is removed according to Method A to form Compound 119.

The phosphonic ester is hydrolyzed according to Example 5 to formCompound 120.

Example 16

BOC-aminopolyethylene glycol- propargyl phenoxy ether was preparedaccording to Method F in 66% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 7.62 m,7.56 m, 6.89 t, 4.70 s, 4.03 t, 3.3-3.7 bm, 3.03 q, 1.37 s. GPC (DMF,PEG standards) M_(n)=7,000; PDI=1.02.

The BOC group is removed according to Method I to form the free aminocompound.

Example 17

BOC-amino-polyethylene glycol-azide (Compound 257) was preparedaccording to Method G followed by Method H in 66% yield. ¹H NMR (400MHz, DMSO-d₆, δ) 6.84 t, 3.3-3.7 bm, 1.37 s. GPC (DMF, PEG standards)M_(n)=7,400; PDI=1.02.

Example 18

TBDMS-PEG-alcohol was prepared according to Method A in 78% yield. ¹HNMR (400 MHz, DMSO-d₆, δ) 4.55 t, 3.3-3.7 bm, 0.83 s, 0.09 s. GPC (DMF,PEG standards) M_(n)=2,400; PDI=1.02.

Example 19

THP-PEG-phosphonic acid was prepared according to Method A. POCl₃ (5equiv) was added and stirred for 6 h at room temperature. The solventwas removed and the residue purified according to Method C giving 62%yield. GPC (DMF, PEG standards) M_(n)=4,100; PDI=1.43.

Example 20

THP-PEG-azide was prepared according to Method G followed by Method H in92% yield. GPC (DMF, PEG standards) M_(n)=2,400; PDI=1.01. ¹H NMR (400MHz, DMSO-d₆, δ) 3.3-3.7 bm, 1.71 m, 1.60 m, 1.44 m. 1.18 m.

Example 21

Oxazoline-PEG-OH was prepared according to Method A followed by Method Bin 49% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 4.14 t, 3.3-3.7 bm, 2.24 t,1.75 quint. GPC (DMF, PEG standards) M_(n)=4,850; PDI=1.04.

Example 22

Carboxylic acid-PEG-OH was prepared according to Method J in 82% yield.¹H NMR (400 MHz, DMSO-d₆, δ) 4.55 t, 3.3-3.7 bm, 2.24 t, 2.13 t, 1.71quint.

Example 23

Oxazoline-PEG-Oxanorbornene was prepared by Method F in 76% yield. ¹HNMR (400 MHz, DMSO-d₆, δ) 6.59 s, 5.12 s, 4.15 t, 3.3-3.7 bm, 2.94 s,2.22 t, 1.75 quint. GPC (DMF, PEG standards) M_(n)=5,100; PDI=1.04.

Example 24

Carboxylic acid-PEG-oxanorbornene was prepared according to Method J in90% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 6.59 s, 5.12 s, 3.3-3.7 bm, 2.94s, 2.24 t, 2.13 t, 1.71 quint. GPC (DMF, PEG standards) M_(n)=5,100;PDI=1.04.

Example 25

Trifluoroacetamide-PEG-alcohol was prepared according to Method K in 73%yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 4.55 t, 3.3-3.7 bm. GPC (DMF, PEGstandards) M_(n)=5,000; PDI=1.07.

Example 26

Azido-PEG-alcohol was prepared according to Method L in 84% yield. ¹HNMR (400 MHz, DMSO-d₆, δ) 4.55 t, 3.3-3.7 bm. GPC (DMF, PEG standards)M_(n)=5,200; PDI=1.03.

Example 27

Propargyl-PEG-alcohol was prepared according to Method L in 87% yield.NMR (400 MHz, DMSO-d₆, δ) 4.55 t, 4.14 d, 3.3-3.7 bm. GPC (DMF, PEGstandards) M_(n)=5,400; PDI=1.03.

Example 28

THP-PEG-oxanorbornene was prepared according to Method F in 97% yield.¹H NMR (400 MHz, DMSO-d₆, δ) 6.55 s, 5.12 s, 4.57 t, 3.3-3.7 bm, 2.92 s,1.71 m, 1.60 m, 1.44 m. 1.18 m.

Example 29

Alcohol-PEG-oxanorbornene was prepared according to Method L in 55%yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 6.55 s, 5.12 s, 4.55 t, 3.3-3.7 bm,2.94 s.

Example 30

Carboxylic acid-PEG-maleimide (compound 18) was prepared according toMethod L in 90% yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 11.94 bs, 7.02 s,3.3-3.7 bm, 2.24 t, 1.70 t. GPC (DMF, PEG standards) M_(n)=2,200;PDI=1.05.

Example 31

NHS-Ester-PEG-maleimide was prepared by dissolving PEG (1 equiv) andN-hydroxysuccinimide (5 equiv) in methylene chloride (˜10 mL/g PEG). DCC(5 equiv) was then added and the solution stirred at room temperaturefor 12 hours. The solution was filtered and the solvent removed. Theresidue was dissolved in isopropanol and precipitated into diethylether, filtered, redissolved in isopropanol and precipitated again intodiethyl ether. A white powder was isolated following filtration in 60%yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 7.02 s, 3.3-3.7 bm, 2.81 s, 2.70 t,1.84 t. GPC (DMF, PEG standards) M_(n)=2,600; PDI=1.05.

Example 32

Azido-PEG-amine hydrochloride was prepared according to Method I in 88%yield. ¹H NMR (400 MHz, DMSO-d₆, δ) 7.86, 3.3-3.7, 2.71.

Example 33

Azido-PEG-amine

Boc-PEG-azide (5 g) was dissolved in methanol (50 mL) and stirred atroom temperature until homogeneous. Anhydrous HCl was bubbled throughthe reaction for 5 minutes and the reaction stirred for an additional 15minutes. The solvent was evaporated, dissolved in saturated NaHCO₃ (30mL, aqueous), then the product extracted with dichloromethane (3×60 mL).The combined organic layers were dried over MgSO₄, and filtered. Thesolvent was removed and the resulting liquid was diluted with a minimalamount of methanol and precipitated in to diethyl ether. A white powder(4.5 g, 90% yield) was isolated following filtration. ¹H NMR (400 MHz,DMSO-d₆, δ) 3.3-3.7, 2.71. IR (cm⁻¹) 3383, 2781, 2102, 1466, 1341, 1279,1241, 1145, 1101, 1060, 956, 841.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1.-27. (canceled)
 28. A compound selected from any of the following,wherein each n is independently 10-2500:

# R^(a) R^(b) 291

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